U.S. patent number 11,136,597 [Application Number 15/434,978] was granted by the patent office on 2021-10-05 for compositions for enhancing targeted gene editing and methods of use thereof.
This patent grant is currently assigned to Carnegie Mellon University, Yale University. The grantee listed for this patent is Carnegie Mellon University, Yale University. Invention is credited to Raman Bahal, Peter Glazer, Danith H. Ly, Nicole Ali McNeer, Elias Quijano, W. Mark Saltzman.
United States Patent |
11,136,597 |
Saltzman , et al. |
October 5, 2021 |
Compositions for enhancing targeted gene editing and methods of use
thereof
Abstract
Compositions and methods for enhancing targeted gene editing and
methods of use thereof are disclosed. In the most preferred
embodiments, gene editing is carried out utilizing a gene editing
composition such as triplex-forming oligonucleotides, CRISPR, zinc
finger nucleases, TALENS, or others, in combination with a gene
modification potentiating agent such as stem cell factor (SCF), a
CHK1 or ATR inhibitor, or a combination thereof. A particular
preferred gene editing composition is triplex-forming peptide
nucleic acids (PNAs) substituted at the .gamma. position for
increased DNA binding affinity. Nanoparticle compositions for
intracellular delivery of the gene editing composition are also
provided and particular advantageous for use with in vivo
applications.
Inventors: |
Saltzman; W. Mark (New Haven,
CT), Glazer; Peter (Guilford, CT), Bahal; Raman
(Hamden, CT), McNeer; Nicole Ali (Westport, CT), Ly;
Danith H. (Pittsburgh, PA), Quijano; Elias (New Haven,
CT) |
Applicant: |
Name |
City |
State |
Country |
Type |
Yale University
Carnegie Mellon University |
New Haven
Pittsburgh |
CT
PA |
US
US |
|
|
Assignee: |
Yale University (New Haven,
CT)
Carnegie Mellon University (Pittsburgh, PA)
|
Family
ID: |
58314495 |
Appl.
No.: |
15/434,978 |
Filed: |
February 16, 2017 |
Prior Publication Data
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Document
Identifier |
Publication Date |
|
US 20170283830 A1 |
Oct 5, 2017 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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62295789 |
Feb 16, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/111 (20130101); C12N 15/102 (20130101); C07K
14/003 (20130101); A61K 48/005 (20130101); A61P
7/00 (20180101); C12N 15/11 (20130101); A61K
9/1647 (20130101); A61P 17/00 (20180101); A61P
31/18 (20180101); C12N 15/907 (20130101); C12N
2310/15 (20130101); C12N 2310/3181 (20130101); C12N
2310/351 (20130101); C12N 15/113 (20130101); C12N
2310/334 (20130101); C12N 2310/152 (20130101); C12N
2310/318 (20130101); C12N 2310/3519 (20130101) |
Current International
Class: |
C07H
21/04 (20060101); C12N 15/11 (20060101); C12N
15/10 (20060101); C07K 14/00 (20060101); C12N
15/90 (20060101); A61K 9/16 (20060101); A61K
48/00 (20060101); C12N 15/113 (20100101) |
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|
Primary Examiner: Chong; Kimberly
Attorney, Agent or Firm: PABST Patent Group LLP
Government Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with government support under AI112443
awarded by National Institutes of Health and under 1012467 awarded
by National Science Foundation. The government has certain rights
in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of and priority to U.S. Ser.
No. 62/295,789 filed Feb. 16, 2016 and which is incorporated by
reference in its entirety.
Claims
We claim:
1. A method of modifying the genomes of CD117+ cells comprising
contacting the CD117+ cells with an effective amount of (i) a gene
editing potentiating agent selected from the group consisting of
receptor tyrosine kinase C-kit ligands, ATR-Chk1 cell cycle
checkpoint pathway inhibitors, and heat shock protein 90 inhibitors
(HSP90i), and (ii) a gene editing technology that can induce
genomic modification through a mechanism comprising a DNA repair
pathway endogenous to the CD117+ cells, to modify the genomes of
the CD117+ cells contacted with both (i) and (ii) at a higher
frequency than an equivalent population of cells contacted with
(ii) in the absence of (i).
2. The method of claim 1 further comprising contacting the cells
with a donor oligonucleotide comprising a sequence that corrects a
mutation(s) in the cells' genomes by insertion or recombination
induced or enhanced by the gene editing technology.
3. The method of claim 2, wherein the cells' genomes have a
mutation underlying a disease or disorder.
4. The method of claim 3, wherein the disease is a
globinopathy.
5. The method of claim 3, wherein the disease is a lysosomal
storage disease.
6. The method of claim 1 further comprising contacting the cells
with a donor oligonucleotide comprising a sequence that induces a
mutation(s) in the cells' genomes by insertion or recombination
induced or enhanced by the gene editing technology.
7. The method of claim 2, wherein the cells are hematopoietic stem
cells.
8. The method of claim 2, wherein the contacting occurs in vivo
following administration of (i), (ii), and the donor
oligonucleotide to a subject in need thereof.
9. The method of claim 8, wherein the subject has a disease or
disorder.
10. The method of claim 8, wherein (i), (ii), the donor
oligonucleotide or a combination thereof are packaged together or
separately in nanoparticles.
11. The method of claim 10, wherein the nanoparticles comprise
poly(lactic-co-glycolic acid) (PLGA).
12. The method of claim 1, wherein the gene editing potentiating
agent is a receptor tyrosine kinase C-kit ligand.
13. A method of modifying the genomes of CD117+ cells comprising
contacting the CD117+ cells with an effective amount of (i) a gene
editing potentiating agent selected from the group consisting of
receptor tyrosine kinase C-kit ligands, ATR-Chk1 cell cycle
checkpoint pathway inhibitors, and heat shock protein 90 inhibitors
(HSP90i), and (ii) a triplex forming composition comprising a
peptide nucleic acid (PNA), wherein one or more of the PNA monomers
is a .gamma.PNA, to modify the genomes of the cells contacted with
both (i) and (ii) at a higher frequency than an equivalent
population of cells contacted with (ii) in the absence of (i).
14. An isolated population of cells treated according to the method
of claim 2.
15. The method of claim 1, wherein the gene editing technology is a
triplex forming molecule.
16. The method of claim 15, wherein the triplex forming molecule
comprises a peptide nucleic acid (PNA) comprising: (a) a Hoogsteen
binding PNA segment; (b) a Watson-Crick binding PNA segment; and
(c) a .gamma.PNA monomer.
17. The method of claim 16, wherein (a) and (b) are linked by a
linker.
18. The method of claim 16, wherein the PNA comprises a
polyethylene glycol moiety.
19. The method of claim 16, wherein the triplex forming molecule is
a tail-clamp PNA.
20. The method of claim 16, wherein the Hoogsteen binding segment
comprises a chemically modified cytosine.
21. The method of claim 15, wherein the cells comprise a genome
encoding a human beta-globin gene, the triplex forming molecule
forms a triplex at the cells' genomic beta-globin locus and, the
triplex forming molecule comprises a Hoogsteen binding peptide
nucleic acid (PNA) segment and a Watson-Crick binding PNA segment,
wherein (i) the Hoogsteen binding segment comprises the sequence
JTTTJTTTJTJT (SEQ ID NO:30) and the Watson-Crick binding segment
comprises the sequence TCTCTTTCTTTC (SEQ ID NO:22) or
TCTCTTTCTTTCAGGGCA (SEQ ID NO:23); (ii) the Hoogsteen binding
segment comprises the sequence TTTTJJJ (SEQ ID NO:31) and the
Watson-Crick binding segment comprises the sequence CCCTTTT (SEQ ID
NO:25) or CCCTTTTGCTAATCATGT (SEQ ID NO:26); (iii) the Hoogsteen
binding segment comprises the sequence TTTJTJJ (SEQ ID NO:32) and
the Watson-Crick binding segment comprises the sequence CCTCTTT
(SEQ ID NO:28) or CCTCTTTGCACCATTCT (SEQ ID NO:29); (iv) the
Hoogsteen binding segment comprises the sequence TJTTTTJTTJ (SEQ ID
NO:36) and the Watson-Crick binding segment comprises the sequence
CTTCTTTTCT (SEQ ID NO:37); (v) the Hoogsteen binding segment
comprises the sequence TTJTTJTTTJ (SEQ ID NO:38) and the
Watson-Crick binding segment comprises the sequence CTTTCTTCTT (SEQ
ID NO:39); (vi) the Hoogsteen binding segment comprises the
sequence JJJTJJTTJT (SEQ ID NO:40) and the Watson-Crick binding
segment comprises TCTTCCTCCC (SEQ ID NO:41); (vii) the Hoogsteen
binding segment comprises the sequence JJTJTTJ (SEQ ID NO:56) and
the Watson-Crick binding segment comprises the sequence CTTCTCC
(SEQ ID NO:46) or CTTCTCCAAAGGAGT (SEQ ID NO:47) or
CTTCTCCACAGGAGTCAG (SEQ ID NO:48) or CTTCTCCACAGGAGTCAGGTGC (SEQ ID
NO:205); (viii) the Hoogsteen binding segment comprises the
sequence TTJJTJT (SEQ ID NO:214) and the Watson-Crick binding
segment comprises the sequence TCTCCTT (SEQ ID NO:50) or
TCTCCTTAAACCTGT (SEQ ID NO:51) or TCTCCTTAAACCTGTCTT (SEQ ID
NO:212); or (ix) the Hoogsteen binding segment comprises the
sequence TJTJTTJT (SEQ ID NO:215) and the Watson-Crick binding
segment comprises the sequence TCTTCTCT (SEQ ID NO:53) or
TCTTCTCTGTCTCCAC (SEQ ID NO:54) or TCTTCTCTGTCTCCACAT (SEQ ID
NO:55); wherein "J" is pseudoisocytosine, and wherein the two
segments are optionally linked by a linker.
22. The method of 21, wherein the triplex forming molecule
comprises a sequence selected from: TABLE-US-00026 (i) (SEQ ID NO:
33) lys-lys-lys-JTTTJTTTJTJT-OOO-TCTCTTTCTTTCAGGGCA- lys-lys-lys;
(ii) (SEQ ID NO: 34)
lys-lys-lys-TTTTJJJ-OOO-CCCTTTTGCTAATCATGT-lys- lys-lys; (iii) (SEQ
ID NO: 35) lys-lys-lys-TTTJTJJ-OOO-CCTCTTTGCACCATTCT-lys- lys-lys,
(iv) (SEQ ID NO: 42)
lys-lys-lys-TJTTTTJTTJ-OOO-CTTCTTTTCT-lys-lys-lys (IVS2-24); (v)
(SEQ ID NO: 43) lys-lys-lys-TTJTTJTTTJ-OOO-CTTTCTTCTT-lys-lys-lys
(IVS2-512); (vi) (SEQ ID NO: 44)
lys-lys-lys-JJJTJJTTJT-OOO-TCTTCCTCCC-lys-lys-lys (IVS2-830); (vii)
(SEQ ID NO: 160) lys-lys-lys-JJTJTTJ-OOO-CTTCTCCAAAGGAGT-lys-lys-
lys; (viii) (SEQ ID NO: 57)
lys-lys-lys-TTJJTJT-OOO-TCTCCTTAAACCTGT-lys-lys- lys; (ix) (SEQ ID
NO: 213) lys-lys-lys-TTJJTJT-OOO-TCTCCTTAAACCTGTCTT-lys- lys-lys
(x) (SEQ ID NO: 58) lys-lys-lys-TJTJTTJT-OOO-TCTTCTCTGTCTCCAC-lys-
lys-lys (tc816); (xi) (SEQ ID NO: 59)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys; (xii) (SEQ
ID NO: 59) lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys
(SCD-tcPNA 1A); (xiii) (SEQ ID NO: 59)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys (SCD-tcPNA
1B); (xiv) (SEQ ID NO: 59)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys (SCD-tcPNA
1C); (xv) (SEQ ID NO: 209)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC- lys-lys-lys
(SCD-tcPNA 1D); (xvi) (SEQ ID NO: 209)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC- lys-lys-lys
(SCD-tcPNA 1E); (xvii) (SEQ ID NO: 209)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC- lys-lys-lys
(SCD-tcPNA 1F); (xviii) (SEQ ID NO: 60)
lys-lys-lys-TJTJTTJT-OOO-TCTTCTCTGTCTCCACAT-lys- lys-lys;
wherein each "lys" is the amino acid lysine, each "J" is
pseudoisocytosine, each "0" is selected from
8-amino-3,6-dioxaoctanoic acid, 6-aminohexanoic acid, and
8-amino-2,6,10-trioxaoctanoic acid, and the bolded and underlined
residues are miniPEG-containing .gamma.PNA residues.
23. The method of claim 15, wherein the cells comprise a genome
encoding a human cystic fibrosis transmembrane conductance
regulator (CFTR) gene, the triplex forming molecule forms a triplex
at the genomic locus of the CFTR gene and the triplex forming
molecule comprises a Hoogsteen binding peptide nucleic acid (PNA)
segment and a Watson-Crick binding PNA segment, wherein (i) the
Hoogsteen binding segment comprises the sequence JTTJJTJTTT (SEQ ID
NO:106) and the Watson-Crick binding segment comprises the sequence
TTTCTCCTTC (SEQ ID NO:98) or TTTCTCCTTCAGTGTTCA (SEQ ID NO:99);
(ii) the Hoogsteen binding segment comprises the sequence TTTTJJT
(SEQ ID NO:107) and the Watson-Crick binding segment comprises the
sequence TCCTTTT (SEQ ID NO:101) or TCCTTTTGCTCACCTGTGGT (SEQ ID
NO:102); (iii) the Hoogsteen binding segment comprises the sequence
TJTTTTTTJJ (SEQ ID NO:108) and the Watson-Crick binding segment
comprises the sequence CCTTTTTTCT (SEQ ID NO:104) or
CCTTTTTTCTGGCTAAGT (SEQ ID NO:105); (iv) the Hoogsteen binding
segment comprises the sequence TJTTTTT (SEQ ID NO:118) Watson-Crick
binding segment comprises the sequence TTTTTCT (SEQ ID NO:111) or
TTTTTCTGTAATTTTTAA (SEQ ID NO:112); (v) the Hoogsteen binding
segment comprises the sequence TJTJTTTJT (SEQ ID NO:119) and the
Watson-Crick binding segment comprises the sequence TCTTTCTCT (SEQ
ID NO:114) or TCTTTCTCTGCAAACTT (SEQ ID NO:115); or (vi) the
Hoogsteen binding segment comprises the sequence TTTJTTT (SEQ ID
NO:120) and the Watson-Crick binding segment comprises the sequence
TTTCTTT (SEQ ID NO:116) or TTTCTTTAAGAACGAGCA (SEQ ID NO:117);
wherein "J" is pseudoisocytosine, and wherein the two segments are
optionally linked by a linker.
24. The method of claim 23, wherein the triplex forming molecule
comprises a sequence selected from: TABLE-US-00027 (i)
lys-lys-lys-JTTJJTJTTT-OOO-TTTCTCCTTCAGTGTTCA-lys-lys-lys; (SEQ ID
NO: 155) (ii)
lys-lys-lys-TTTTJJT-OOO-TCCTTTTGCTCACCTGTGGT-lys-lys-lys; (SEQ ID
NO: 156) (iii)
lys-lys-lys-TJTTTTTTJJ-OOO-CCTTTTTTCTGGCTAAGT-lys-lys-lys; (SEQ ID
NO: 157) (iv)
lys-lys-lys-TJTTTTT-OOO-TTTTTCTGTAATTTTTAA-lys-lys-lys; (SEQ ID NO:
121) (v) lys-lys-lys-TJTJTTTJT-OOO-TCTTTCTCTGCAAACTT-lys-lys-lys;
(SEQ ID NO: 122) (vi)
lys-lys-lys-TTTJTTT-OOO-TTTCTTTAAGAACGAGCA-lys-lys-lys; and (SEQ ID
NO: 123) (vii)
lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ ID NO:
93)
wherein each "lys" is the amino acid lysine, each "J" is
pseudoisocytosine, each "0" is selected from
8-amino-3,6-dioxaoctanoic acid, 6-aminohexanoic acid, and
8-amino-2,6,10-trioxaoctanoic acid, and the bolded and underlined
residues are miniPEG-containing .gamma.PNA residues.
25. The method of claim 3, wherein the disease is cystic
fibrosis.
26. The method of claim 1, wherein DNA repair pathway is the
homology-dependent repair (HDR) pathway.
27. The method of claim 1, wherein the gene editing technology
comprises an enzyme that induces a single or double strand break in
cells' genomes.
28. The method of claim 27, wherein the enzyme is a Cas
endonuclease, zinc finger nuclease (ZFN), transcription
activator-like effector nucleases (TALEN), or intron encoded
meganuclease.
29. The method of claim 13, further comprising contacting the cells
with a donor oligonucleotide comprising a sequence that introduces
or corrects a mutation(s) in the cells' genomes by insertion or
recombination induced or enhanced by the triplex forming
composition.
30. The method of claim 13, wherein the gene editing potentiating
agent is a receptor tyrosine kinase C-kit ligand.
Description
REFERENCE TO SEQUENCE LISTING
The Sequence Listing submitted as a text file named
"YU_6876_5_ST25.txt," created on Nov. 3, 2020, and having a size of
93,350 bytes is hereby incorporated by reference pursuant to 37
C.F.R .sctn. 1.52(e)(5).
FIELD OF THE INVENTION
The field of the invention is generally related to gene editing
technology used in combination with a gene modification
potentiating agent, and compositions and methods of use thereof for
ex vivo and in vivo gene editing.
BACKGROUND OF THE INVENTION
Gene editing in hematopoietic stem/progenitor cells (HSPCs)
provides an attractive strategy for treatment of inherited
disorders such as sickle cell anemia and .beta.-thalassemia. Genes
can be selectively edited by several methods, including targeted
nucleases such as zinc finger nucleases (ZFNs) (Haendel, et al.,
Gene Ther., 11:28-37 (2011)) and CRISPRs (Yin, et al., Nat.
Biotechnol., 32:551-553 (2014)), short fragment homologous
recombination (SFHR) (Goncz, et al., Oligonucleotides, 16:213-224
(2006)), or triplex-forming oligonucleotides (TFOs) (Vasquez, et
al., Science, 290:530-533 (2000)). Recent excitement has focused on
CRISPR/Cas9 technology because of its ease of use and facile
reagent design (Doudna, et al., Science, 346:1258096 (2014)).
However, like ZFNs, the CRISPR approach introduces an active
nuclease into cells, which can lead to off-target cleavage in the
genome (Cradick, et al., Nucleic Acids Res., 41:9584-9592 (2013)),
a problem that so far has not been eliminated.
One alternative is triplex-forming peptide nucleic acid (PNA)
oligomers designed to bind site-specifically to genomic DNA via
strand invasion and formation of PNA/DNA/PNA triplexes via both
Watson-Crick and Hoogsteen binding) with a displaced DNA strand
(Egholm, et al., Nature (London), 365:566-568 (1993); Nielsen, et
al., Science (Washington, D.C., 1883-), 254:1497-1500 (1991);
Faruqi, et al., Proc Natl Acad Sci USA, 95:1398-1403 (1998)). PNAs
have a charge-neutral peptide-like backbone and nucleobases
enabling hybridization with DNA and RNA with high affinity.
PNA/DNA/PNA triplexes recruit the cell's endogenous DNA repair
systems to initiate site-specific modification of the genome when
single-stranded "donor DNAs" are co-delivered as templates
containing the desired sequence modification (Rogers, et al., Proc.
Natl. Acad. Sci. USA, 99:16695-16700 (2002)).
PNA-induced genome modification is believed to be mediated in part
by the nucleotide excision repair (NER) and homology-dependent
repair (HDR) pathways (Rogers, et al., Proc. Natl. Acad. Sci. USA,
99:16695-16700 (2002); Chin, et al., Molecular Carcinogenesis,
48:389-399 (2009)). Both NER and HDR are high fidelity pathways,
and the PNAs lack any intrinsic nuclease activity. Together these
features may account for the very low frequencies of off-target
genotoxicity seen with PNA-mediated gene editing compared to
nuclease based approaches (McNeer, et al., Gene Therapy, 20:658-669
(2013); Schleifinan, et al., Chem. Biol. (Cambridge, Mass., U.S.),
18:1189-1198 (2011); Schleifman, et al., Mol. Ther.--Nucleic Acids,
2:e135 (2013)). Tail-clamp PNAs (tcPNAs) with an extended
Watson-Crick binding domain can enhance gene editing in human
hematopoietic cells with increased efficiency and specificity
(Schleifman, et al., Chem. Biol. (Cambridge, Mass., U.S.),
18:1189-1198 (2011)) and that polymer nanoparticles (NPs) can
effectively deliver these molecules into human HSPCs both ex vivo
and in vivo in a humanized mouse model (McNeer, et al., Gene
Therapy, 20:658-669 (2013); Bahal, et al., Curr. Gene Ther.,
14:331-342 (2014)).
Nonetheless, compositions and methods for improved gene editing are
needed.
It is an object of the invention to provide potentiating agents
that increase gene modification induced or enhanced by gene editing
technology.
It is another object of the invention to provide triplex forming
molecules with enhanced DNA binding.
It is a further object of the invention to provide gene
modification formulations that achieve therapeutically significant
target site modification with reduced low off-target
modification.
SUMMARY OF THE INVENTION
Highly elevated levels of gene editing in hematopoietic
stem/progenitor cells are achieved using triplex-forming peptide
nucleic acids (PNAs) substituted at the .gamma. position for
increased DNA binding affinity in combination with stimulation of
the stem cell factor (SCF)/c-Kit pathway. The SCF/c-Kit pathway is
believed to boost DNA repair gene expression and homology-dependent
repair activity as evidence shows that stimulation is correlated
with elevated DNA repair, specifically increased HDR activity and
increased levels of HDR gene expression, including BRCA2 and Rad51.
In a mouse model of human .beta.-thalassemia, injection with SCF
plus nanoparticles containing .gamma.PNAs and donor DNAs yielded
amelioration of the disease phenotype, with clinically relevant
.beta.-globin gene correction frequencies (4% in bone marrow) and
extremely low off-target effects. The mice showed alleviation of
anemia with sustained elevation of blood hemoglobin levels into the
normal range, reduced reticulocyte counts, and reversal of
splenomegaly.
Compositions and methods for enhancing targeted gene editing and
methods of use thereof are disclosed. In the most preferred
embodiments, gene editing is carried out utilizing a gene editing
composition such as triplex-forming oligonucleotides, CRISPR, zinc
finger nucleases, TALENS, or others, in combination with a gene
modification potentiating agent such as SCF, a CHK1 or ATR
inhibitor, a DNA polymerase alpha inhibitor, a heat shock protein
90 inhibitor (HSP90i) or a combination thereof. A particularly
preferred gene editing composition is triplex-forming peptide
nucleic acids (PNAs) substituted at the .gamma. position for
increased DNA binding affinity. Nanoparticle compositions for
intracellular delivery of the gene editing composition are also
provided and particularly advantageous for use with in vivo
applications.
For example, an exemplary method of modifying the genome of a cell
can include contacting the cell with an effective amount of (i) a
gene editing potentiating agent selected from the group consisting
of tyrosine kinase C-kit ligands, ATR-Chk1 cell cycle checkpoint
pathway inhibitors, DNA polymerase alpha inhibitors, and heat shock
protein 90 inhibitors (HSP90i), and (ii) a gene editing technology
that can induce genomic modification of the cell selected from the
group consisting of triplex forming molecules, pseudocomplementary
oligonucleotides, a CRISPR system, zinc finger nucleases (ZFN), and
transcription activator-like effector nucleases (TALEN); wherein
genomic modification occurs at a higher frequency in a population
of cells contacted with both (i) and (ii), then in an equivalent
population contacted with (ii) in the absence of (i). The method
can further include contacting the cells with a donor
oligonucleotide including, for example, a sequence that corrects or
induces a mutation(s) in the cell's genome by insertion or
recombination of the donor induced or enhanced by the gene editing
technology.
A preferred C-kit ligand is a stem cell factor protein or fragment
thereof sufficient to cause dimerization of C-kit and activate its
tyrosine kinase activity. In some embodiments, the C-kit ligand is
a nucleic acid such as an mRNA or an expression vector encoding a
stem cell factor protein or fragment thereof sufficient to cause
dimerization of C-kit and activate its tyrosine kinase
activity.
ATR-Chk1 cell cycle checkpoint pathway inhibitors are typically
small molecules though they can also be inhibitory nucleic acids
such as siRNA that target and reduce expression a gene in the
pathway. Inhibitors include, for example, AZD7762,
SCH900776/MK-8776, IC83/LY2603618, LY2606368, GDC-0425,
PF-00477736, XL844, CEP-3891, SAR-020106, CCT-244747, Arry-575,
SB218075, Schisandrin B, NU6027, NVP-BEZ235, VE-821, VE-822
(VX-970), AZ20, AZD6738, MIRIN, KU5593, VE-821, NU7441, LCA, and
L189.
In some embodiments, the cell's genome has a mutation underlying a
disease or disorder, for example a genetic disorder such as
hemophilia, globinopathies, cystic fibrosis, xeroderma pigmentosum,
muscular dystrophy, and lysosomal storage diseases. The
globinopathy can be sickle cell anemia or beta-thalassemia. The
lysosomal storage disease can be Gaucher's disease, Fabry disease,
or Hurler syndrome. In some embodiments, the method induces a
mutation that reduces HIV infection, for example, by reducing an
activity of a cell surface receptor that facilitates entry of HIV
into the cell.
The contacting of the compositions with the cell can occur ex vivo.
In some embodiments, the ex vivo-treated cells are hematopoietic
stem cells. The modified cells can be administered to a subject in
need thereof in an effective amount to treat one or more symptoms
of a disease or disorder such as hemophilia, a globinopathy, cystic
fibrosis, xeroderma pigmentosum, muscular dystrophy, a lysosomal
storage disease, or HIV.
In vivo applications are also provided. For example in some
embodiments, the potentiating agent, gene editing technology and
optionally the donor oligonucleotide are administered to a subject
in need thereof. Each of the foregoing can be in the same or
different pharmaceutical compositions and can be administered to
the subject in any order. In preferred embodiments, the
compositions induce or enhance in vivo gene modification in an
effective amount to reduce one or more symptoms of the disease or
disorder, for example, hemophilia, a globinopathy, xeroderma
pigmentosum, a lysosomal storage disease, or HIV in the
subject.
Any of the disclosed compositions including potentiating agent,
gene editing technology, and/or donor oligonucleotide can be
packaged together or separately in nanoparticles. In preferred
embodiments, the nanoparticles include poly(lactic-co-glycolic
acid) (PLGA) alone or in a blend with poly(beta-amino) esters
(PBAEs). In particular embodiments, the nanoparticles include a
blend of PLGA and PBAE having between about 10 and about 20 percent
PBAE (wt %). In preferred embodiments, the nanoparticles are
prepared by double emulsion. In some embodiments the gene editing
technology, the donor oligonucleotide or a combination thereof are
complexed with a polycation prior to preparation of the
nanoparticles.
Functional molecules such as targeting moieties, a cell penetrating
peptides, or a combination thereof can be associated with, linked,
conjugated, or otherwise attached directly or indirectly to the
potentiating agent, the gene editing technology, the nanoparticle,
or a combination thereof. In particularly preferred embodiments, a
cell penetrating peptide including the sequence GALFLGFLGAAGSTMGAWS
QPKKKRKV (SEQ ID NO:12) (MPG (Synthetic chimera: SV40 Lg T.
Ant.+HIV gb41 coat)) is conjugated to the surface of the
nanoparticles.
Improved DNA-binding triplex forming molecules are also provided.
The triplex forming molecules can be utilized in all manners of
gene modification including those methods both with and without a
potentiating agent. The triplex forming composition typically
includes a Hoogsteen binding peptide nucleic acid (PNA) segment and
a Watson-Crick binding PNA segment collectively totaling no more
than about 50 nucleobases in length, wherein the two segments can
bind or hybridize to a target region having a polypurine stretch in
a cell's genome to induce strand invasion, displacement, and
formation of a triple-stranded molecule among the two PNA segments
and the polypurine stretch. The Hoogsteen binding segment binds to
the target duplex by Hoogsteen binding for a length of at least
five nucleobases, and the Watson-Crick binding segment typically
binds to the target duplex by Watson-Crick binding for a length of
least five nucleobases.
In preferred embodiments, one or more of the PNA monomers are
.gamma.PNA. The side chain at the .gamma. position of the
.gamma.PNA monomer(s) can be, for example, the side chain of an
amino acid selected from the group consisting of alanine, serine,
threonine, cysteine, valine, leucine, isoleucine, methionine,
proline, phenylalanine, tyrosine, aspartic acid, glutamic acid,
asparagine, glutamine, histidine, lysine, arginine, and the
derivatives thereof. In some embodiments, the side chain at the
.gamma. position of the .gamma.PNA monomer(s) is a diethylene
glycol ("miniPEG"). In some embodiments, all of the peptide nucleic
acid monomers in the Hoogsteen-binding portion only, all of the
peptide nucleic acid monomers in the Watson-Crick-binding portion
only, or all of the peptide nucleic monomers in the PNA oligomer
are .gamma.PNA monomers. In some embodiments, alternating residues
in the Hoogsteen-binding portion only, the Watson-Crick-binding
portion only, or across the entire PNA are PNA and .gamma.PNA.
Specific exemplary sequences are provided below.
In some embodiments, one or more of the cytosines is replaced with
a clamp-G (9-(2-guanidinoethoxy) phenoxazine). In preferred
embodiments, the Hoogsteen binding segment includes one or more
chemically modified cytosines selected from the group consisting of
pseudocytosine, pseudoisocytosine, and 5-methylcytosine. The
Watson-Crick binding segment preferably includes a tail sequence of
up to fifteen nucleobases that binds to the target duplex by
Watson-Crick binding outside of the triplex. In preferred
embodiments, the two segments are linked by a linker, for example,
between 1 and 10 units of 8-amino-3,6-dioxaoctanoic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic showing a strategy for targeted correction
of a .beta.-globin gene IVS2-654 (C.fwdarw.T) mutation in
.beta.-globin/GFP transgenic mice using triplex-forming tail clamp
PNAs (tcPNAs) and donor DNAs. FIG. 1B is an illustration showing
tcPNA and .gamma.tcPNA oligomers (SEQ ID NOS:33-35, 162, and 158,
respectively) designed to bind to the homopurine regions within
intron 2 of the human .beta.-globin gene in the vicinity of the
thalassemia-associated mutation IVS2-654 (C.fwdarw.T), and a
scrambled control sequence (SEQ ID NO:158). FIG. 1C is the chemical
structures of DNA, unmodified PNA and miniPEG gamma PNA
(.sup.MP.gamma.PNA) units. FIG. 1D is a bar graph showing gene
correction of the IVS2-654 (C.fwdarw.T) mutation within the
.beta.-globin/GFP fusion gene in mouse bone marrow cells treated ex
vivo with blank NPs and NPs containing donor DNA (SEQ ID NO:65)
alone or in combination with tcPNA3 (SEQ ID NO:35), tcPNA2 (SEQ ID
NO:34), or tcPNA1 (SEQ ID NO:33). The % GFP+ cells among mouse bone
marrow cells was determined by flow cytometry and indicates
successful gene editing. Data are shown as mean.+-.s.e., n=3;
statistical analysis was performed with student's t-test, asterisk,
p<0.05. FIG. 1E is a line graph showing release of total nucleic
acids (PNAs in combination with donor DNA (SEQ ID NO:65):
.gamma.tcPNA4 (SEQ ID NO:162), tcPNA1 (SEQ ID NO:33), tcPNA2 (SEQ
ID NO:34), tcPNA3 (SEQ ID NO:35) or .gamma.tcPNA4-Scr (SEQ ID
NO:158); or DNA donor (SEQ ID NO:65) alone) from PLGA nanoparticles
during incubation at 37.degree. C. in PBS. At 64 hrs, the residual
nucleic acid in the NP pellet was extracted and the total nucleic
acid load was calculated as a sum of absorbance obtained from the
pellet and supernatant. FIG. 1F is a bar graph showing % GFP+ cells
determined by flow cytometry among mouse bone marrow cells (from
.beta.-globin/GFP transgenic mice) after ex vivo treatment with
PLGA NPs containing tcPNA1 (SEQ ID NO:33), .gamma.tcPNA4 (SEQ ID
NO:162), or .gamma.tcPNA4-Scr (SEQ ID NO:158) plus donor DNAs (SEQ
ID NO:65). Replicates and statistics as above for FIG. 1D. FIG. 1G
is a bar graph showing mouse total bone marrow cells were treated
with either blank NPs or NPs containing .gamma.tcPNA4 (SEQ ID
NO:162) and donor DNA (SEQ ID NO:65) and were plated for a
colony-forming cell assay in methylcellulose medium with selected
cytokines for growth of granulocyte/macrophage colonies (CFU-G,
CFU-M and CFU-GM) or combined colonies (CFU-GEMM, granulocyte,
erythroid, monocyte/macrophage, megakaryocyte. Numbers of each type
of colony per 300,000 plated cells are shown. Data are shown as
mean.+-.s.d., n=3. FIG. 1H is a bar graph showing the results of a
comet assay to measure DNA breaks in NP-treated bone marrow cells.
Cells were treated with NPs containing either tcPNA1/donor DNA (SEQ
ID NOS:33 and 65), .gamma.tcPNA4/donor DNA (SEQ ID NOS:162 and 65),
or bleomycin/donor DNA (SEQ ID NO:65), as indicated. DNA tail
moment provides a measurement of the extent of breaks. Data are
shown as mean.+-.s.e., n=3.
FIG. 2A is a bar graph showing % GFP expression in treated mouse
bone marrow cells based on selected hematopoietic cell surface
markers. Total bone marrow was treated with NPs containing either
tcPNA1/donor DNA (SEQ ID NOS:33 and 65) or .gamma.tcPNA4/donor DNA
(SEQ ID NOS:162 and 65), and then the cells were stained using
antibodies specific for the indicated markers and assayed by flow
cytometry for marker and GFP expression. Data are shown as
mean.+-.s.e., n=3; statistical analysis was performed with
student's t-test, asterisk, p<0.05. FIG. 2B is a bar graph
showing % GFP expressing CD117 (c-Kit+) cells after ex vivo
treatment with NPs carrying .gamma.tcPNAs and donor DNAs (SEQ ID
NOS:162 and 65) versus with blank NPs. Data are shown as
mean.+-.s.e., n=3; statistical analysis was performed with
student's t-test, asterisk, p<0.05. FIG. 2C is a bar graph
showing % GFP expressing CD117+ cells from .beta.-globin/GFP
transgenic mice after ex vivo treatment with NPs containing
.gamma.tcPNA4/donor DNA (SEQ ID NOS:162 and 65) with or without
prior treatment with the c-Kit ligand, SCF. Data are shown as
mean.+-.s.e., n=3; statistical analysis was performed with
student's t-test, asterisk, p<0.05. FIG. 2D is a bar graph
showing % GFP expressing CD117+ cells isolated from
.beta.-globin/GFP transgenic mice after ex vivo treatment with NPs
containing .gamma.tcPNA4/donor DNA (SEQ ID NOS:162 and 65) in the
presence or absence of selected c-Kit pathway kinase inhibitors:
dasatinib (inhibits c-Kit), MEK162 (inhibits mitogen/extracellular
signal-regulated kinase, MEK) and BKM120 (inhibits
phosphatidylinositol-3-kinase, PI3K). Data are shown as
mean.+-.s.e., n=3; statistical analysis was performed with
student's t-test, asterisk, p<0.05. FIGS. 2E and 2F are bar
graphs showing qPCR determination of mRNA expression levels of
BRCA2 (2E) and Rad51 (2I) in CD117- and CD117+ cells. FIG. 2G is a
heat map showing up-regulated genes involved in DNA repair pathways
in CD117+ cells with or without treatment with SCF; rows are
clustered by Euclidean distance measure. FIG. 2H is a bar graph
showing the results of a gene assay for homology-dependent repair
(HDR) activity in the presence or absence of selected c-Kit pathway
kinase inhibitors: dasatinib (inhibits c-Kit), MEK162 (inhibits
mitogen/extracellular signal-regulated kinase, MEK) and BKM120
(inhibits phosphatidylinositol-3-kinase, PI3K). Inset shows a
diagram of the luciferase reporter gene assay for repair of a
nuclease-induced double-strand break by homology-dependent repair
(HDR). Luciferase expression occurs only after homologous
recombination and is scored as % reactivation of the DSB-damaged
plasmid, normalized to a transfection control. FIG. 2I is a bar
graph showing the results of an HDR assay in CD117+ cells with or
without the addition of SCF. Data are shown as mean.+-.s.e., n=3;
statistical analysis was performed with student's t-test, asterisk,
p<0.05. FIG. 2J is a bar graph showing the results of an HDR
assay in DLD-1 cells either proficient or deficient in the homology
dependent repair factor BRCA2 as a validation of the assay. Data
are shown as mean.+-.s.e., n=3; statistical analysis was performed
with student's t-test, asterisk, p<0.05.
FIGS. 3A and 3B are dot plots showing frequencies of gene editing
(GFP expression) in bone marrow (3A) and spleen (3B) cells from
.beta.-globin/GFP transgenic mice (6 mice per group) injected or
not (as indicated) with 15.6 .mu.g of SCF i.p. followed by a single
treatment of 4 mg of NPs injected intravenously. Each group
received either blank NPs or NPs containing .gamma.tcPNA4 and donor
DNA (SEQ ID NOS:162 and 65), with or without SCF and were harvested
and analysed two days later. Each data point represents analysis of
cells from a single mouse. Statistical analyses were performed
using student's t-test: asterisk, p<0.05. FIG. 3C is a bar graph
showing the results of deep-sequencing analysis to quantify the
frequency of targeted gene editing (% modification frequency
IVS2-654 (T.fwdarw.C)) in vivo in CD117+ cells from bone marrow and
spleen of .beta.-globin/GFP mice treated as described for FIGS. 3A
and 3B. Error bars indicate standard error of proportions.
FIGS. 4A-4C are line graphs showing blood hemoglobin levels (g/dl)
of thalassemic mice treated with blank NPs, SCF plus scrambled
.gamma.tcPNA4-Scr/donor DNA (SEQ ID NOS:158 and 65) NPs, or with
SCF plus .gamma.tcPNA4/donor DNA (SEQ ID NOS:162 and 65) NPs
performed at the indicated times after treatment. Each line
represents an individual mouse followed over time. FIG. 4D is a bar
graph showing reticulocyte counts (% of total RBCs) calculated in
blood smears from thalassemic mice treated with either blank NPs or
with NPs containing .gamma.tcPNA4/donor DNA (SEQ ID NOS:162 and 65)
plus SCF on days 0 and 36 post treatment. FIG. 4E is a bar graphs
showing the % gene modification (T.fwdarw.C) as determined by
deep-sequencing analysis of genomic DNA from bone marrow cells
after treatment of thalassemic mice with either blank NPs or with
NPs containing and .gamma.tcPNA4/donor DNA (SEQ ID NOS:162 and 65)
plus SCF.
FIG. 5A is a flow diagram illustrating a GFP/beta globin gene
correction assay. FIG. 5B is a bar graph showing gene correction of
cells treated with nanoparticles containing tcPNA1 (SEQ ID NO:191)
and donor DNA (SEQ ID NO:65) alone, or in combination with an
ataxia telangiectasia and Rad3-related protein (ATR) pathway
inhibitor (MIRIN, KU5593, VE-821, NU7441, LCA, or L189). FIG. 5C is
a bar graph showing gene correction of cells treated with
nanoparticles containing tcPNA1 (SEQ ID NO:191) and donor DNA (SEQ
ID NO:65) alone, or in combination with a Checkpoint Kinase 1
inhibitor (Chk1i) (SB218075), a DNA polymerase alpha inhibitor
(Aphi) (aphidicolin) or a polyADP ribose polymerase (PARPi)
(AZD-2281 (olaparib)). FIG. 5D is a bar graph showing gene
correction of control (blank), and cells treated with nanoparticles
containing tcPNA1 (SEQ ID NO:191) and donor DNA (SEQ ID NO:76)
alone, or in combination with a heat shock protein 90 inhibitor
(HSP90i) (STA-9090 (ganetespib)).
FIG. 6A is an illustration of a Sickle Cell Disease mutation
(GAG.fwdarw.GTG) in the human beta globin gene, relative to the ATG
transcriptional start site and exemplary tcPNAs. FIG. 6B shows the
sequences of exemplary PNAs: tcPNA1:
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCAAAGGAGT-lys-lys-lys (SEQ ID NO:66);
tcPNA2: lys-lys-lys-TTJJTJT-OOO-TCTCCTTAAACCTGT-lys-lys-lys (SEQ ID
NO:67); and tcPNA3:
lys-lys-lys-TJTJTTJT-OOO-TCTTCTCTGTCTCCAC-lys-lys-lys (SEQ ID
NO:68). FIG. 6C shows the sequence of a DNA donor (SEQ ID
NO:64).
FIG. 7A is a bar graph showing the results of a MQAE
(N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide) assay
(delta(AFU)/(delta(Time (sec)) measuring chloride flux for negative
control CFBE cells; CFBE cells treated with blank nanoparticles,
PNA2: lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ
ID NO:93)-loaded nanoparticles, PNA2 (SEQ ID NO:93)-loaded
nanoparticles with an MPG peptide, .gamma.PNA2
lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ ID
NO:69)-loaded nanoparticles; and untreated positive control
wildtype 16HBE14o-cells. FIG. 7B is a dot pot showing nasal
potential difference (NPD) (pretreatment, after treatment with
.gamma.PNA2 (SEQ ID NO:69)-loaded nanoparticles, and after
treatment with blank nanoparticles) measured using a non-invasive
assay used to detect chloride potential differences in vivo.
FIG. 8A is an illustration of a mutation (G.fwdarw.A) in the CFTR
gene (W1282X) relative to three exemplary tcPNAs. FIG. 8B provides
the sequences of the tcPNAs: CF-1236
lys-lys-lys-JTTJJTJTTT-OOO-TTTCTCCTTCAGTGTTCA-lys-lys-lys (SEQ ID
NO:169), CF-1314
lys-lys-lys-TTTTJJT-OOO-TCCTTTTGCTCACCTGTGGT-lys-lys-lys (SEQ ID
NO:170), and CF-1329:
lys-lys-lys-TJTTTTTTJJ-OOO-CCTTTTTTCTGGCTAAGT-lys-lys-lys (SEQ ID
NO:171). FIG. 8C provides the sequence of an exemplary donor DNA:
T(s)C(s)T(s)TGGGATTCAATAACCTTGCAGACAGTGGAGGAAGGCCTT
TGGCGTGATACCACAGG-(s)T(s)G(s) (SEQ ID NO:109).
FIG. 9A is an illustration of a mutation (G.fwdarw.T) in the CFTR
gene (G542X) relative to three exemplary tcPNAs. FIG. 9B provides
the sequences of the tcPNAs: CF-302
lys-lys-lys-TJTTTTT-OOO-TTTTTCTGTAATTTTTAA-lys-lys-lys (SEQ ID
NO:172), CF-529
lys-lys-lys-TJTJTTTJT-OOO-TCTTTCTCTGCAAACTT-lys-lys-lys (SEQ ID
NO:173), and CF-586
lys-lys-lys-TTTJTTT-OOO-TTTCTTTAAGAACGAGCA-lys-lys-lys (SEQ ID
NO:174). FIG. 9C provides the sequence of an exemplary donor DNA:
T(s)C(s)C(s)-AAGTTTGCAGAGAAAGATAATATAGTCCTTGGAGAAGGAGGAATCA
CCCTGAGTGGA-G(s)G(s)T(s) (SEQ ID NO:124).
FIG. 10A is an illustration of Strategy for targeted correction of
a .beta.-globin gene containing SCD mutation (A.fwdarw.T) mutation
and tcPNAs designed to bind to homopurine regions near the
mutation. FIGS. 10B-10C are bar graphs showing hydrodynamic
diameter of formulated PLGA nanoparticles measured using dynamic
light scattering in PBS buffer (FIG. 10B) and zeta potential of
formulated PLGA nanoparticles (FIG. 10C). Data in both graphs are
presented as mean.+-.s.e.m., n=3. FIGS. 10D-10E are bar graphs
showing the results of deep-sequencing analysis to quantify the
frequency of targeted gene editing in vivo in bone marrow cells of
Berkley "Berk" mice (FIG. 10D) and Townes mice (FIG. 10E). Error
bars indicate standard error of proportions.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used herein, "affinity tags" are defined herein as molecular
species which form highly specific, non-covalent, physiochemical
interactions with defined binding partners. Affinity tags which
form highly specific, non-covalent, physiochemical interactions
with one another are defined herein as "complementary".
As used herein, "coupling agents" are defined herein as molecular
entities which associate with polymeric nanoparticles and provide
substrates that facilitate the modular assembly and disassembly of
functional elements onto the nanoparticle. Coupling agents can be
conjugated to affinity tags. Affinity tags allow for flexible
assembly and disassembly of functional elements which are
conjugated to affinity tags that form highly specific, noncovalent,
physiochemical interactions with affinity tags conjugated to
adaptor elements. Coupling agents can also be covalently coupled to
functional elements in the absence of affinity tags.
As used herein, the term "isolated" describes a compound of
interest (e.g., either a polynucleotide or a polypeptide) that is
in an environment different from that in which the compound
naturally occurs, e.g., separated from its natural milieu such as
by concentrating a peptide to a concentration at which it is not
found in nature. "Isolated" is meant to include compounds that are
within samples that are substantially enriched for the compound of
interest and/or in which the compound of interest is partially or
substantially purified.
As used herein with respect to nucleic acids, the term "isolated"
includes any non-naturally-occurring nucleic acid sequence, since
such non-naturally-occurring sequences are not found in nature and
do not have immediately contiguous sequences in a
naturally-occurring genome.
As used herein, the term "host cell" refers to prokaryotic and
eukaryotic cells into which a nucleic acid can be introduced.
As used herein, "transformed" and "transfected" encompass the
introduction of a nucleic acid into a cell by one of a number of
techniques known in the art.
As used herein, the phrase that a molecule "specifically binds" to
a target refers to a binding reaction which is determinative of the
presence of the molecule in the presence of a heterogeneous
population of other biologics. Thus, under designated immunoassay
conditions, a specified molecule binds preferentially to a
particular target and does not bind in a significant amount to
other biologics present in the sample. Specific binding of an
antibody to a target under such conditions requires the antibody be
selected for its specificity to the target. A variety of
immunoassay formats may be used to select antibodies specifically
immunoreactive with a particular protein. For example, solid-phase
ELISA immunoassays are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See, e.g.,
Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, for a description of immunoassay
formats and conditions that can be used to determine specific
immunoreactivity. Specific binding between two entities means an
affinity of at least 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9, or
10.sup.10 M.sup.-1. Affinities greater than 10.sup.8M.sup.-1 are
preferred.
As used herein, "targeting molecule" is a substance which can
direct a nanoparticle to a receptor site on a selected cell or
tissue type, can serve as an attachment molecule, or serve to
couple or attach another molecule. As used herein, "direct" refers
to causing a molecule to preferentially attach to a selected cell
or tissue type. This can be used to direct cellular materials,
molecules, or drugs, as discussed below.
As used herein, the terms "antibody" or "immunoglobulin" are used
to include intact antibodies and binding fragments thereof.
Typically, fragments compete with the intact antibody from which
they were derived for specific binding to an antigen fragment
including separate heavy chains, light chains Fab, Fab' F(ab')2,
Fabc, and Fv. Fragments are produced by recombinant DNA techniques,
or by enzymatic or chemical separation of intact immunoglobulins.
The term "antibody" also includes one or more immunoglobulin chains
that are chemically conjugated to, or expressed as, fusion proteins
with other proteins. The term "antibody" also includes a bispecific
antibody. A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp.
Immunol., 79:315-321 (1990); Kostelny, et al., J. Immunol., 148,
1547-1553 (1992). As used herein, the terms "epitope" or "antigenic
determinant" refer to a site on an antigen to which B and/or T
cells respond. B-cell epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
and more usually, at least 5 or 8-10, amino acids, in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include, for example, x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn
E. Morris, Ed. (1996). Antibodies that recognize the same epitope
can be identified in a simple immunoassay showing the ability of
one antibody to block the binding of another antibody to a target
antigen. T-cells recognize continuous epitopes of about nine amino
acids for CD8 cells or about 13-15 amino acids for CD4 cells. T
cells that recognize the epitope can be identified by in vitro
assays that measure antigen-dependent proliferation, as determined
by .sup.3H-thymidine incorporation by primed T cells in response to
an epitope (Burke, et al., J. Inf. Dis., 170:1110-19 (1994)), by
antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges, et
al., J. Immunol., 156, 3901-3910) or by cytokine secretion.
As used herein, the term "small molecule," as used herein,
generally refers to an organic molecule that is less than about
2000 g/mol in molecular weight, less than about 1500 g/mol, less
than about 1000 g/mol, less than about 800 g/mol, or less than
about 500 g/mol. Small molecules are non-polymeric and/or
non-oligomeric.
As used herein, the term "carrier" or "excipient" refers to an
organic or inorganic ingredient, natural or synthetic inactive
ingredient in a formulation, with which one or more active
ingredients are combined.
As used herein, the term "pharmaceutically acceptable" means a
non-toxic material that does not interfere with the effectiveness
of the biological activity of the active ingredients.
As used herein, the terms "effective amount" or "therapeutically
effective amount" means a dosage sufficient to alleviate one or
more symptoms of a disorder, disease, or condition being treated,
or to otherwise provide a desired pharmacologic and/or physiologic
effect. The precise dosage will vary according to a variety of
factors such as subject-dependent variables (e.g., age, immune
system health, etc.), the disease or disorder being treated, as
well as the route of administration and the pharmacokinetics of the
agent being administered.
As used herein, the term "prevention" or "preventing" means to
administer a composition to a subject or a system at risk for or
having a predisposition for one or more symptom caused by a disease
or disorder to cause cessation of a particular symptom of the
disease or disorder, a reduction or prevention of one or more
symptoms of the disease or disorder, a reduction in the severity of
the disease or disorder, the complete ablation of the disease or
disorder, stabilization or delay of the development or progression
of the disease or disorder.
II. Gene Editing Potentiating Factors
It has been discovered that certain potentiating factors can be
used to increase the efficacy of gene editing technologies. Gene
expression profiling on SCF-treated CD117+ cells versus untreated
CD117+ cells discussed in the Examples below showed additional
up-regulation of numerous DNA repair genes including RAD51 and
BRCA2. These results and others discussed below indicate that a
functional c-Kit signaling pathway mediates increased HDR and
promotes gene editing, rather than CD117 simply being a phenotypic
marker. When CD117+ cells were treated with SCF, expression of
these DNA repair genes was increased even more, correlating with a
further increase in gene editing.
Accordingly, compositions and methods of increasing the efficacy of
gene editing technology are provided. As used herein a "gene
editing potentiating factor" or "gene editing potentiating agent"
or "potentiating factor or "potentiating agent" refers a compound
that increases the efficacy of editing (e.g., mutation, including
insertion, deletion, substitution, etc.) of a gene, genome, or
other nucleic acid) by a gene editing technology relative to use of
the gene editing technology in the absence of the compound.
Preferred gene editing technologies suitable for use alone or more
preferably in combination with the disclosed potentiating factors
are discussed in more detail below. In certain preferred
embodiments, the gene editing technology is a triplex-forming
.gamma.PNA and donor DNA, optionally, but preferably in a
nanoparticle composition.
Potentiating factors include, for example, DNA damage or
repair-stimulating or -potentiating factors. Preferably the factor
is one that engages one or more endogenous high fidelity DNA repair
pathways. In some embodiments, the factor is one that increases
expression of Rad51, BRCA2, or a combination thereof.
As discussed in more detail below, the preferred methods typically
include contacting cells with an effective amount of a gene editing
potentiating factor. The contacting can occur ex vivo, for example
isolated cells, or in vivo following, for example, administration
of the potentiating factor to a subject.
A. C-Kit Ligands
In some embodiments, the factor is an activator of the receptor
tyrosine kinase c-Kit. CD117 (also known as mast/stem cell growth
factor receptor or proto-oncogene c-Kit protein) is a receptor
tyrosine kinase expressed on the surface of hematopoietic stem and
progenitor cells as well as other cell types. Stem cell factor
(SCF), the ligand for c-Kit, causes dimerization of the receptor
and activates its tyrosine kinase activity to trigger downstream
signaling pathways that can impact survival, proliferation, and
differentiation. SCF and c-Kit are reviewed in Lennartsson and
Ronnstrand, Physiological Reviews, 92(4):1619-1649 (2012)).
The human SCF gene encodes for a 273 amino acid transmembrane
protein, which contains a 25 amino acid N-terminal signal sequence,
a 189 amino acid extracellular domain, a 23 amino acid
transmembrane domain, and a 36 amino acid cytoplasmic domain. A
canonical human SCF amino acid sequence is:
TABLE-US-00001 (SEQ ID NO: 1, UniProtKB-P21583 (SCF_HUMAN))
MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNNKDVTKLVANLPK
DYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNY
SIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNRSI
DAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRNDSS
SSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKR
QPSLTRAVENIQINEEDNEISMLQEKEREFQEV.
The secreted soluble form of SCF is generated by proteolytic
processing of the membrane-anchored precursor. A cleaved, secreted
soluble form of human SCF is underlined in SEQ ID NO:1, which
corresponds to SEQ ID NO:2 without the N-terminal methionine.
TABLE-US-00002 MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISE
MVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKD
LKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPE
KDSRVSVTKPFMLPPVA (SEQ ID NO: 2, Preprotech Recombinant Human SCF
Catalog Number: 300-07).
Murine and rat SCF are fully active on human cells. A canonical
mouse SCF amino acid sequence is:
TABLE-US-00003 (SEQ ID NO: 3, UniProtKB-P20826 (SCF_MOUSE))
MKKTQTWIITCIYLQLLLENPLVKTKEICGNPVTDNVKDITKLVANLPND
YMITLNYVAGMDVLPSHCWLRDMVIQLSLSLTTLLDKFSNISEGLSNYSI
IDKLGKIVDDLVLCMEENAPKNIKESPKRPETRSFTPEEFFSIFNRSIDA
FKDFMVASDTSDCVLSSTLGPEKDSRVSVTKPFMLPPVAASSLRNDSSSS
NRKAAKAPEDSGLQWTAMALPALISLVIGFAFGALYWKKKQSSLTRAVEN
IQINEEDNEISMLQQKEREFQEV.
A cleaved, secreted soluble form of mouse SCF is underlined in SEQ
ID NO:3, which corresponds to SEQ ID NO:4 without the N-terminal
methionine.
TABLE-US-00004 MKEICGNPVTDNVKDITKLVANLPNDYMITLNYVAGMDVLPSHCWLRD
MVIQLSLSLTTLLDKFSNISEGLSNYSIIDKLGKIVDDLVLCMEENAPKN
IKESPKRPETRSFTPEEFFSIFNRSIDAFKDFMVASDTSDCVLSSTLGPE
KDSRVSVTKPFMLPPVA (SEQ ID NO: 4, Preprotech Recombinant Murine SCF
Catalog Number: 250-03)
A canonical mouse SCF amino acid sequence is:
TABLE-US-00005 (SEQ ID NO: 5, UniProtKB-P21581 (SCF_RAT))
MKKTQTWIITCIYLQLLLFNPLVKTQEICRNPVTDNVKDITKLVANLPND
YMITLNYVAGMDVLPSHCWLRDMVTHLSVSLTTLLDKFSNISEGLSNYS
IIDKLGKIVDDLVACMEENAPKNVKESLKKPETRNFTPEEFFSIFNRSID
AFKDFMVASDTSDCVLSSTLGPEKDSRVSVTKPFMLPPVAASSLRNDSSS
SNRKAAKSPEDPGLQWTAMALPALISLVIGFAFGALYWKKKQSSLTRAV
ENIQINEEDNEISMLQQKEREFQEV.
A cleaved, secreted soluble form of rat SCF is underlined in SEQ ID
NO:5, which corresponds to SEQ ID NO:6 without the N-terminal
methionine.
TABLE-US-00006 MQEICRNPVTDNVKDITKLVANLPNDYMITLNYVAGMDVLPSHCWLRD
MVTHLSVSLTTLLDKFSNISEGLSNYSIIDKLGKIVDDLVACMEENAPKN
VKESLKKPETRNFTPEEFFSIFNRSIDAFKDFMVASDTSDCVLSSTLGPE
KDSRVSVTKPFMLPPVA (SEQ ID NO: 6, Shenandoah Biotechnology, Inc.,
Recombinant Rat SCF (Stem Cell Factor) Catalog Number: 300-32).
In some embodiments, the factor is a SCF such as any of SEQ ID
NO:1-6, with or without the N-terminal methionine, or a functional
fragment thereof, or a variant thereof with at least 60, 65, 70,
75, 80, 85, 90, 95, 96, 97, 98, 99, or more sequence identity to
any one of SEQ ID NO:1-6.
It will be appreciated that SCF can be administered to cells or a
subject as SCF protein, or as a nucleic acid encoding SCF
(transcribed RNA, DNA, DNA in an expression vector). Accordingly,
nucleic acid sequences, including RNA (e.g., mRNA) and DNA
sequences, encoding SEQ ID NOS:1-6 are also provided, both alone
and inserted into expression cassettes and vectors. For example, a
sequence encoding SCF can be incorporated into an autonomously
replicating plasmid, a virus (e.g., a retrovirus, lentivirus,
adenovirus, or herpes virus), or into the genomic DNA of a
prokaryote or eukaryote.
The observed effect of SCF indicates that other cytokines or growth
factors including, but not limited to, erythropoietin, GM-CSF, EGF
(especially for epithelial cells; lung epithelia for cystic
fibrosis), hepatocyte growth factor etc., could similarly serve to
boost gene editing potential in bone marrow cells or in other
tissues. In some embodiments, gene editing is enhanced in specific
cell types using cytokines targeted to these cell types.
B. Replication Modulators
In some embodiments, the potentiating factor is a replication
modulator that can, for example, manipulate replication progression
and/or replication forks. For example, the ATR-Chk1 cell cycle
checkpoint pathway has numerous roles in protecting cells from DNA
damage and stalled replication, one of the most prominent being
control of the cell cycle and prevention of premature entry into
mitosis (Thompson and Eastman, Br J Clin Pharmacol., 76(3): 358-369
(2013), Smith, et al., Adv Cancer Res., 108:73-112 (2010)).
However, Chk1 also contributes to the stabilization of stalled
replication forks, the control of replication origin firing and
replication fork progression, and homologous recombination. DNA
polymerase alpha also known as Pol .alpha. is an enzyme complex
found in eukaryotes that is involved in initiation of DNA
replication. Hsp90 (heat shock protein 90) is a chaperone protein
that assists other proteins to fold properly, stabilizes proteins
against heat stress, and aids in protein degradation.
Experimental results show that inhibitors of CHK1 and ATR in the
DNA damage response pathway, as well as DNA polymerase alpha
inhibitors and HSP90 inhibitors, substantially boost gene editing
by triplex-forming PNAs and single-stranded donor DNA
oligonucleotides. Accordingly, in some embodiments, the
potentiating factor is a CHK1 or ATR pathway inhibitor, a DNA
polymerase alpha inhibitor, or an HSP90 inhibitor. The inhibitor
can be a functional nucleic acid, for example siRNA, miRNA,
aptamers, ribozymes, triplex forming molecules, RNAi, or external
guide sequences that targets CHK1, ATR, or another molecule in the
ATR-Chk1 cell cycle checkpoint pathway; DNA polymerase alpha; or
HSP90 and reduces expression or active of ATR, CHK1, DNA polymerase
alpha, or HSP90.
Preferably, the inhibitor is a small molecule. For example, the
potentiating factor can be a small molecule inhibitor of ATR-Chk1
Cell Cycle Checkpoint Pathway Inhibitor. Such inhibitors are known
in the art, and many have been tested in clinical trials for the
treatment of cancer. Exemplary CHK1 inhibitors include, but are not
limited to, AZD7762, SCH900776/MK-8776, IC83/LY2603618, LY2606368,
GDC-0425, PF-00477736, XL844, CEP-3891, SAR-020106, CCT-244747,
Arry-575 (Thompson and Eastman, Br J Clin Pharmacol., 76(3):
358-369 (2013)), and SB218075. Exemplary ATR pathway inhibitors
include, but are not limited to Schisandrin B, NU6027, NVP-BEZ235,
VE-821, VE-822 (VX-970), AZ20, AZD6738, MIRIN, KU5593, VE-821,
NU7441, LCA, and L189 (Weber and Ryan, Pharmacology &
Therapeutics, 149:124-138 (2015)).
In some embodiments, the potentiating factor is a DNA polymerase
alpha inhibitor, such as aphidicolin.
In some embodiments, the potentiating factor is a heat shock
protein 90 inhibitor (HSP90i) such as STA-9090 (ganetespib). Other
HSP90 inhibitors are known in the art and include, but are not
limited to, benzoquinone ansamycin antibiotics such as geldanamycin
(GA); 17-AAG (17-Allylamino-17-demethoxy-geldanamycin); 17-DMAG
(17-dimethylaminoethylamino-17-demethoxy-geldanamycin)
(Alvespimycin); IPI-504 (Retaspimycin); and AUY922 (Tatokoro, et
al., EXCLI J., 14:48-58 (2015)).
III. Gene Editing Technology
Gene editing technologies can be used alone or preferably in
combination with a potentiating agent. Exemplary gene editing
technologies include, but are not limited to, triplex-forming,
pseudocomplementary oligonucleotides, CRISPR/Cas, zinc finger
nucleases, and TALENs, each of which are discussed in more detail
below. As discussed in more detail below, some gene editing
technologies are used in combination with a donor oligonucleotide.
In some embodiments, the gene editing technology is the donor
oligonucleotide, which can be used be used alone to modify genes.
Strategies include, but are not limited to, small fragment
homologous replacement (e.g., polynucleotide small DNA fragments
(SDFs)), single-stranded oligodeoxynucleotide-mediated gene
modification (e.g., ssODN/SSOs) and other described in Sargent,
Oligonucleotides, 21(2): 55-75 (2011)), and elsewhere. Other
suitable gene editing technologies include, but are not limited to
intron encoded meganucleases that are engineered to change their
target specificity. See, e.g., Arnould, et al., Protein Eng. Des.
Sel., 24(1-2):27-31 (2011)).
A. Triplex-Forming Molecules
1. Compositions
Compositions containing "triplex-forming molecules," that bind to
duplex DNA in a sequence-specific manner to form a triple-stranded
structure include, but are not limited to, triplex-forming
oligonucleotides (TFOs), peptide nucleic acids (PNA), and "tail
clamp" PNA (tcPNA). The triplex-forming molecules can be used to
induce site-specific homologous recombination in mammalian cells
when combined with donor DNA molecules. The donor DNA molecules can
contain mutated nucleic acids relative to the target DNA sequence.
This is useful to activate, inactivate, or otherwise alter the
function of a polypeptide or protein encoded by the targeted duplex
DNA. Triplex-forming molecules include triplex-forming
oligonucleotides and peptide nucleic acids. Triplex forming
molecules are described in U.S. Pat. Nos. 5,962,426, 6,303,376,
7,078,389, 7,279,463, 8,658,608, U.S. Published Application Nos.
2003/0148352, 2010/0172882, 2011/0268810, 2011/0262406,
2011/0293585, and published PCT application numbers WO 1995/001364,
WO 1996/040898, WO 1996/039195, WO 2003/052071, WO 2008/086529, WO
2010/123983, WO 2011/053989, WO 2011/133802, WO 2011/13380, Rogers,
et al., Proc Natl Acad Sci USA, 99:16695-16700 (2002), Majumdar, et
al., Nature Genetics, 20:212-214 (1998), Chin, et al., Proc Natl
Acad Sci USA, 105:13514-13519 (2008), and Schleifman, et al., Chem
Biol., 18:1189-1198 (2011). As discussed in more detail below,
triplex forming molecules are typically single-stranded
oligonucleotides that bind to polypyrimidine:polypurine target
motif in a double stranded nucleic acid molecule to form a
triple-stranded nucleic acid molecule. The single-stranded
oligonucleotide typically includes a sequence substantially
complementary to the polypurine strand of the
polypyrimidine:polypurine target motif.
a. Triplex-Forming Oligonucleotides (TFOs)
Triplex-forming oligonucleotides (TFOs) are defined as
oligonucleotides which bind as third strands to duplex DNA in a
sequence specific manner. The oligonucleotides are synthetic or
isolated nucleic acid molecules which selectively bind to or
hybridize with a predetermined target sequence, target region, or
target site within or adjacent to a human gene so as to form a
triple-stranded structure.
Preferably, the oligonucleotide is a single-stranded nucleic acid
molecule between 7 and 40 nucleotides in length, most preferably 10
to 20 nucleotides in length for in vitro mutagenesis and 20 to 30
nucleotides in length for in vivo mutagenesis. The base composition
may be homopurine or homopyrimidine. Alternatively, the base
composition may be polypurine or polypyrimidine. However, other
compositions are also useful.
The oligonucleotides are preferably generated using known DNA
synthesis procedures. In one embodiment, oligonucleotides are
generated synthetically. Oligonucleotides can also be chemically
modified using standard methods that are well known in the art.
The nucleotide sequence of the oligonucleotides is selected based
on the sequence of the target sequence, the physical constraints
imposed by the need to achieve binding of the oligonucleotide
within the major groove of the target region, and the need to have
a low dissociation constant (K.sub.d) for the
oligonucleotide/target sequence. The oligonucleotides have a base
composition which is conducive to triple-helix formation and is
generated based on one of the known structural motifs for third
strand binding. The most stable complexes are formed on
polypurine:polypyrimidine elements, which are relatively abundant
in mammalian genomes. Triplex formation by TFOs can occur with the
third strand oriented either parallel or anti-parallel to the
purine strand of the duplex. In the anti-parallel, purine motif,
the triplets are G.G:C and A.A:T, whereas in the parallel
pyrimidine motif, the canonical triplets are C.sup.+.G:C and T.A:T.
The triplex structures are stabilized by two Hoogsteen hydrogen
bonds between the bases in the TFO strand and the purine strand in
the duplex. A review of base compositions for third strand binding
oligonucleotides is provided in U.S. Pat. No. 5,422,251.
Preferably, the oligonucleotide binds to or hybridizes to the
target sequence under conditions of high stringency and
specificity. Most preferably, the oligonucleotides bind in a
sequence-specific manner within the major groove of duplex DNA.
Reaction conditions for in vitro triple helix formation of an
oligonucleotide probe or primer to a nucleic acid sequence vary
from oligonucleotide to oligonucleotide, depending on factors such
as oligonucleotide length, the number of G:C and A:T base pairs,
and the composition of the buffer utilized in the hybridization
reaction. An oligonucleotide substantially complementary, based on
the third strand binding code, to the target region of the
double-stranded nucleic acid molecule is preferred.
As used herein, a triplex forming molecule is said to be
substantially complementary to a target region when the
oligonucleotide has a heterocyclic base composition which allows
for the formation of a triple-helix with the target region. As
such, an oligonucleotide is substantially complementary to a target
region even when there are non-complementary bases present in the
oligonucleotide. As stated above, there are a variety of structural
motifs available which can be used to determine the nucleotide
sequence of a substantially complementary oligonucleotide.
b. Peptide Nucleic Acids (PNA)
In another embodiment, the triplex-forming molecules are peptide
nucleic acids (PNAs). Peptide nucleic acids are molecules in which
the phosphate backbone of oligonucleotides is replaced in its
entirety by repeating N-(2-aminoethyl)-glycine units and
phosphodiester bonds are replaced by peptide bonds. The various
heterocyclic bases are linked to the backbone by methylene carbonyl
bonds. PNAs maintain spacing of heterocyclic bases that are similar
to oligonucleotides, but are achiral and neutrally charged
molecules. Peptide nucleic acids are comprised of peptide nucleic
acid monomers. The heterocyclic bases can be any of the standard
bases (uracil, thymine, cytosine, adenine and guanine) or any of
the modified heterocyclic bases described below.
PNAs can bind to DNA via Watson-Crick hydrogen bonds, but with
binding affinities significantly higher than those of a
corresponding nucleotide composed of DNA or RNA. The neutral
backbone of PNAs decreases electrostatic repulsion between the PNA
and target DNA phosphates. Under in vitro or in vivo conditions
that promote opening of the duplex DNA, PNAs can mediate strand
invasion of duplex DNA resulting in displacement of one DNA strand
to form a D-loop.
Highly stable triplex PNA:DNA:PNA structures can be formed from a
homopurine DNA strand and two PNA strands. The two PNA strands may
be two separate PNA molecules, or two PNA molecules linked together
by a linker of sufficient flexibility to form a single bis-PNA
molecule. In both cases, the PNA molecule(s) forms a triplex
"clamp" with one of the strands of the target duplex while
displacing the other strand of the duplex target. In this
structure, one strand forms Watson-Crick base pairs with the DNA
strand in the anti-parallel orientation (the Watson-Crick binding
portion), whereas the other strand forms Hoogsteen base pairs to
the DNA strand in the parallel orientation (the Hoogsteen binding
portion). A homopurine strand allows formation of a stable
PNA/DNA/PNA triplex. PNA clamps can form at shorter homopurine
sequences than those required by triplex-forming oligonucleotides
(TFOs) and also do so with greater stability.
Suitable molecules for use in linkers of bis-PNA molecules include,
but are not limited to, 8-amino-3,6-dioxaoctanoic acid, referred to
as an O-linker, and 6-aminohexanoic acid. Poly(ethylene) glycol
monomers can also be used in bis-PNA linkers. A bis-PNA linker can
contain multiple linker molecule monomers in any combination.
PNAs can also include other positively charged moieties to increase
the solubility of the PNA and increase the affinity of the PNA for
duplex DNA. Commonly used positively charged moieties include the
amino acids lysine and arginine, although other positively charged
moieties may also be useful. Lysine and arginine residues can be
added to a bis-PNA linker or can be added to the carboxy or the
N-terminus of a PNA strand.
c. Tail Clamp Peptide Nucleic Acids (tcPNA)
Although polypurine:polypyrimidine stretches do exist in mammalian
genomes, it is desirable to target triplex formation in the absence
of this requirement. In some embodiments such as PNA,
triplex-forming molecules include a "tail" added to the end of the
Watson-Crick binding portion. Adding additional nucleobases, known
as a "tail" or "tail clamp", to the Watson-Crick binding portion
that bind to the target strand outside the triple helix further
reduces the requirement for a polypurine:polypyrimidine stretch and
increases the number of potential target sites. The tail is most
typically added to the end of the Watson-Crick binding sequence
furthest from the linker. This molecule therefore mediates a mode
of binding to DNA that encompasses both triplex and duplex
formation (Kaihatsu, et al., Biochemistry, 42(47):13996-4003
(2003); Bentin, et al., Biochemistry, 42(47):13987-95 (2003)). For
example, if the triplex-forming molecules are tail clamp PNA
(tcPNA), the PNA/DNA/PNA triple helix portion and the PNA/DNA
duplex portion both produce displacement of the pyrimidine-rich
strand, creating an altered helical structure that strongly
provokes the nucleotide excision repair pathway and activating the
site for recombination with a donor DNA molecule (Rogers, et al.,
Proc. Natl. Acad. Sci. USA., 99(26):16695-700 (2002)).
Tails added to clamp PNAs (sometimes referred to as bis-PNAs) form
tail-clamp PNAs (referred to as tcPNAs) that have been described by
Kaihatsu, et al., Biochemistry, 42(47):13996-4003 (2003); Bentin,
et al., Biochemistry, 42(47):13987-95 (2003). tcPNAs are known to
bind to DNA more efficiently due to low dissociation constants. The
addition of the tail also increases binding specificity and binding
stringency of the triplex-foiming molecules to the target duplex.
It has also been found that the addition of a tail to clamp PNA
improves the frequency of recombination of the donor
oligonucleotide at the target site compared to PNA without the
tail.
d. PNA Modifications
PNAs can also include other positively charged moieties to increase
the solubility of the PNA and increase the affinity of the PNA for
duplex DNA. Commonly used positively charged moieties include the
amino acids lysine and arginine, although other positively charged
moieties may also be useful. Lysine and arginine residues can be
added to a bis-PNA linker or can be added to the carboxy or the
N-terminus of a PNA strand. Common modifications to PNA are
discussed in Sugiyama and Kittaka, Molecules, 18:287-310 (2013))
and Sahu, et al., J. Org. Chem., 76, 5614-5627 (2011), each of
which are specifically incorporated by reference in their
entireties, and include, but are not limited to, incorporation of
charged amino acid residues, such as lysine at the termini or in
the interior part of the oligomer; inclusion of polar groups in the
backbone, carboxymethylene bridge, and in the nucleobases; chiral
PNAs bearing substituents on the original N-(2-aminoethyl)glycine
backbone; replacement of the original aminoethylglycyl backbone
skeleton with a negatively-charged scaffold; conjugation of high
molecular weight polyethylene glycol (PEG) to one of the termini;
fusion of PNA to DNA to generate a chimeric oligomer, redesign of
the backbone architecture, conjugation of PNA to DNA or RNA. These
modifications improve solubility but often result in reduced
binding affinity and/or sequence specificity. In some embodiments,
the some or all of the PNA monomers are modified at the gamma
position in the polyamide backbone (.gamma.PNAs) as illustrated
below (wherein "B" is a nucleobase and "R" is a substitution at the
gamma position).
##STR00001##
Substitution at the gamma position creates chirality and provides
helical pre-organization to the PNA oligomer, yielding
substantially increased binding affinity to the target DNA
(Rapireddy, et al., Biochemistry, 50(19):3913-8 (2011)). Other
advantageous properties can be conferred depending on the chemical
nature of the specific substitution at the gamma position (the "R"
group in the chiral .gamma.PNA above).
One class of .gamma. substitution, is miniPEG, but other residues
and side chains can be considered, and even mixed substitutions can
be used to tune the properties of the oligomers. "MiniPEG" and "MP"
refers to diethylene glycol. MiniPEG-containing .gamma.PNAs are
conformationally preorganized PNAs that exhibit superior
hybridization properties and water solubility as compared to the
original PNA design and other chiral .gamma.PNAs. .gamma.PNAs
prepared from L-amino acids adopt a right-handed helix, while those
prepared from D-amino acids adopt a left-handed helix; however,
only the right-handed helical .gamma.PNAs hybridize to DNA or RNA
with high affinity and sequence selectivity. In the most preferred
embodiments, some or all of the PNA monomers are miniPEG-containing
.gamma.PNAs (Sahu, et al., J. Org. Chem., 76, 5614-5627 (2011). In
the embodiments, tcPNAs are prepared wherein every other PNA
monomer on the Watson-Crick binding side of the linker is a
miniPEG-containing .gamma.PNA. Accordingly, the tail clamp side of
the PNA has alternating PNA and miniPEG-containing .gamma.PNA
monomers.
In some embodiments PNA-mediated gene editing are achieved via
additional or alternative .gamma. substitutions or other PNA
chemical modifications including but limited to those introduced
above and below. Examples of .gamma. substitution with other side
chains include that of alanine, serine, threonine, cysteine,
valine, leucine, isoleucine, methionine, proline, phenylalanine,
tyrosine, aspartic acid, glutamic acid, asparagine, glutamine,
histidine, lysine, arginine, and the derivatives thereof. The
"derivatives thereof" herein are defined as those chemical moieties
that are covalently attached to these amino acid side chains, for
instance, to that of serine, cysteine, threonine, tyrosine,
aspartic acid, glutamic acid, asparagine, glutamine, lysine, and
arginine.
In addition to .gamma.PNAs showing consistently improved gene
editing potency the level of off-target effects in the genome
remains extremely low. This is in keeping with the lack of any
intrinsic nuclease activity in the PNAs (in contrast to ZFNs or
CRISPR/Cas9 or TALENS), and reflects the mechanism of
triplex-induced gene editing, which acts by creating an altered
helix at the target-binding site that engages endogenous high
fidelity DNA repair pathways. As discussed above, the SCF/c-Kit
pathway also stimulates these same pathways, providing for enhanced
gene editing without increasing off-target risk or cellular
toxicity.
Additionally, any of the triplex forming sequences can be modified
to include guanidine-G-clamp ("G-clamp") PNA monomer(s) to enhance
PNA binding. .gamma.PNAs with substitution of cytosine by clamp-G
(9-(2-guanidinoethoxy) phenoxazine), a cytosine analog that can
form five H-bonds with guanine, and can also provide extra base
stacking due to the expanded phenoxazine ring system and
substantially increased binding affinity. In vitro studies indicate
that a single clamp-G substitution for C can substantially enhance
the binding of a PNA-DNA duplex by 23.degree. C. (Kuhn, et al.,
Artificial DNA, PNA & ANA, 1(1):45-53(2010)). As a result,
.gamma.PNAs containing G-clamp substitutions can have further
increased activity.
The structure of a clamp-G monomer-to-G base pair (clamp-G
indicated by the "X") is illustrated below in comparison to C-G
base pair.
##STR00002##
Some studies have shown improvements using D-amino acids in peptide
synthesis.
2. Triplex-Forming Target Sequence Considerations
The triplex-forming molecules bind to a predetermined target region
referred to herein as the "target sequence," "target region," or
"target site." The target sequence for the triplex-forming
molecules can be within or adjacent to a human gene encoding, for
example the beta globin, cystic fibrosis transmembrane conductance
regulator (CFTR) or other gene discussed in more detail below, or
an enzyme necessary for the metabolism of lipids, glycoproteins, or
mucopolysaccharides, or another gene in need of correction. The
target sequence can be within the coding DNA sequence of the gene
or within an intron. The target sequence can also be within DNA
sequences which regulate expression of the target gene, including
promoter or enhancer sequences or sites that regulate RNA
splicing.
The nucleotide sequences of the triplex-forming molecules are
selected based on the sequence of the target sequence, the physical
constraints, and the need to have a low dissociation constant
(K.sub.d) for the triplex-forming molecules/target sequence. As
used herein, triplex-forming molecules are said to be substantially
complementary to a target region when the triplex-forming molecules
has a heterocyclic base composition which allows for the formation
of a triple-helix with the target region. As such, a
triplex-forming molecules is substantially complementary to a
target region even when there are non-complementary bases present
in the triplex-forming molecules.
There are a variety of structural motifs available which can be
used to determine the nucleotide sequence of a substantially
complementary oligonucleotide. Preferably, the triplex-forming
molecules bind to or hybridize to the target sequence under
conditions of high stringency and specificity. Reaction conditions
for in vitro triple helix formation of an triplex-forming molecules
probe or primer to a nucleic acid sequence vary from
triplex-forming molecules to triplex-forming molecules, depending
on factors such as the length triplex-forming molecules, the number
of G:C and A:T base pairs, and the composition of the buffer
utilized in the hybridization reaction.
a. Target Sequence Considerations for TFOs
Preferably, the TFO is a single-stranded nucleic acid molecule
between 7 and 40 nucleotides in length, most preferably 10 to 20
nucleotides in length for in vitro mutagenesis and 20 to 30
nucleotides in length for in vivo mutagenesis. The base composition
may be homopurine or homopyrimidine. Alternatively, the base
composition may be polypurine or polypyrimidine. However, other
compositions are also useful. Most preferably, the oligonucleotides
bind in a sequence-specific manner within the major groove of
duplex DNA. An oligonucleotide substantially complementary, based
on the third strand binding code, to the target region of the
double-stranded nucleic acid molecule is preferred. The
oligonucleotides will have a base composition which is conducive to
triple-helix formation and will be generated based on one of the
known structural motifs for third strand binding. The most stable
complexes are formed on polypurine:polypyrimidine elements, which
are relatively abundant in mammalian genomes. Triplex formation by
TFOs can occur with the third strand oriented either parallel or
anti-parallel to the purine strand of the duplex. In the
anti-parallel, purine motif, the triplets are G.G:C and A.A:T,
whereas in the parallel pyrimidine motif, the canonical triplets
are C.sup.+.G:C and T.A:T. The triplex structures are stabilized by
two Hoogsteen hydrogen bonds between the bases in the TFO strand
and the purine strand in the duplex. A review of base compositions
for third strand binding oligonucleotides is provided in U.S. Pat.
No. 5,422,251.
The oligonucleotides are preferably generated using known DNA
synthesis procedures. In one embodiment, oligonucleotides are
generated synthetically. Oligonucleotides can also be chemically
modified using standard methods that are well known in the art.
b. Target Sequence Considerations for PNAs
Some triplex-forming molecules, such as PNA and tcPNA invade the
target duplex, with displacement of the polypyrimidine strand, and
induce triplex formation with the polypurine strand of the target
duplex by both Watson-Crick and Hoogsteen binding. Preferably, both
the Watson-Crick and Hoogsteen binding portions of the triplex
forming molecules are substantially complementary to the target
sequence. Although, as with triplex-forming oligonucleotides, a
homopurine strand is needed to allow formation of a stable
PNA/DNA/PNA triplex, PNA clamps can form at shorter homopurine
sequences than those required by triplex-forming oligonucleotides
and also do so with greater stability.
Preferably, PNAs are between 6 and 50 nucleotides in length. The
Watson-Crick portion should be 9 or more nucleobases in length,
optionally including a tail sequence. More preferably, the
Watson-Crick binding portion is between about 9 and 30 nucleobases
in length, optionally including a tail sequence of between 0 and
about 15 nucleobases. More preferably, the Watson-Crick binding
portion is between about 10 and 25 nucleobases in length,
optionally including a tail sequence of between 0 and about 10
nucleobases. In the most preferred embodiment, the Watson-Crick
binding portion is between 15 and 25 nucleobases in length,
optionally including a tail sequence of between 5 and 10
nucleobases. The Hoogsteen binding portion should be 6 or more
nucleobases in length. Most preferably, the Hoogsteen binding
portion is between about 6 and 15 nucleobases, inclusive.
The triplex-forming molecules are designed to target the polypurine
strand of a polypurine:polypyrimidine stretch in the target duplex
nucleotide. Therefore, the base composition of the triplex-forming
molecules may be homopyrimidine. Alternatively, the base
composition may be polypyrimidine. The addition of a "tail" reduces
the requirement for polypurine:polypyrimidine run. Adding
additional nucleobases, known as a "tail," to the Watson-Crick
binding portion of the triplex-forming molecules allows the
Watson-Crick binding portion to bind/hybridize to the target strand
outside the site of polypurine sequence for triplex formation.
These additional bases further reduce the requirement for the
polypurine:polypyrimidine stretch in the target duplex and
therefore increase the number of potential target sites.
Triplex-forming oligonucleotides (TFOs) also require a
polypurine:polypyrimidine sequence to a form a triple helix. TFOs
may require stretch of at least 15 and preferably 30 or more
nucleotides. Peptide nucleic acids require fewer purines to a form
a triple helix, although at least 10 or preferably more may be
needed. Peptide nucleic acids including a tail, also referred to
tail clamp PNAs, or tcPNAs, require even fewer purines to a form a
triple helix. A triple helix may be formed with a target sequence
containing fewer than 8 purines. Therefore, PNAs should be designed
to target a site on duplex nucleic acid containing between 6-30
polypurine:polypyrimidines, preferably, 6-25
polypurine:polypyrimidines, more preferably 6-20
polypurine:polypyrimidines.
The addition of a "mixed-sequence" tail to the Watson-Crick-binding
strand of the triplex-forming molecules such as PNAs also increases
the length of the triplex-forming molecule and, correspondingly,
the length of the binding site. This increases the target
specificity and size of the lesion created at the target site and
disrupts the helix in the duplex nucleic acid, while maintaining a
low requirement for a stretch of polypurine:polypyrimidines.
Increasing the length of the target sequence improves specificity
for the target, for example, a target of 17 base pairs will
statistically be unique in the human genome. Relative to a smaller
lesion, it is likely that a larger triplex lesion with greater
disruption of the underlying DNA duplex will be detected and
processed more quickly and efficiently by the endogenous DNA repair
machinery that facilitates recombination of the donor
oligonucleotide.
The triple-forming molecules are preferably generated using known
synthesis procedures. In one embodiment, triplex-forming molecules
are generated synthetically. Triplex-forming molecules can also be
chemically modified using standard methods that are well known in
the art.
B. Pseudocomplementary Oligonucleotides
The gene editing technology can be pseudocomplementary
oligonucleotides such as those disclosed in U.S. Pat. No.
8,309,356. "Double duplex-forming molecules," are oligonucleotides
that bind to duplex DNA in a sequence-specific manner to form a
four-stranded structure. Double duplex-forming molecules, such as a
pair of pseudocomplementary oligonucleotides, can induce
recombination with a donor oligonucleotide at a chromosomal site in
mammalian cells. Pseudocomplementary oligonucleotides are
complementary oligonucleotides that contain one or more
modifications such that they do not recognize or hybridize to each
other, for example due to steric hindrance, but each can recognize
and hybridize to its complementary nucleic acid strands at the
target site. Preferred pseudocomplementary oligonucleotides include
Pseudocomplementary peptide nucleic acids (pcPNAs). A
pseudocomplementary oligonucleotide is said to be substantially
complementary to a target region when the oligonucleotide has a
base composition which allows for the formation of a double duplex
with the target region. As such, an oligonucleotide is
substantially complementary to a target region even when there are
non-complementary bases present in the oligonucleotide.
This strategy can be more efficient and provides increased
flexibility over other methods of induced recombination such as
triple-helix oligonucleotides and bis-peptide nucleic acids which
prefer a polypurine sequence in the target double-stranded DNA. The
design ensures that the pseudocomplementary oligonucleotides do not
pair with each other but instead bind the cognate nucleic acids at
the target site, inducing the formation of a double duplex.
The predetermined region that the double duplex-forming molecules
bind to can be referred to as a "double duplex target sequence,"
"double duplex target region," or "double duplex target site." The
double duplex target sequence (DDTS) for the double duplex-forming
oligonucleotides can be, for example, within or adjacent to a human
gene in need of induced gene correction. The DDTS can be within the
coding DNA sequence of the gene or within introns. The DDTS can
also be within DNA sequences which regulate expression of the
target gene, including promoter or enhancer sequences.
The nucleotide sequence of the pseudocomplementary oligonucleotides
is selected based on the sequence of the DDTS. Therapeutic
administration of pseudocomplementary oligonucleotides involves two
single stranded oligonucleotides unlinked, or linked by a linker.
One pseudocomplementary oligonucleotide strand is complementary to
the DDTS, while the other is complementary to the displaced DNA
strand. The use of pseudocomplementary oligonucleotides,
particularly pcPNAs are not subject to limitation on sequence
choice and/or target length and specificity as are triplex-forming
oligonucleotides, helix-invading peptide nucleic acids (bis-PNAs)
and side-by-side minor groove binders. Pseudocomplementary
oligonucleotides do not require third-strand Hoogsteen-binding, and
therefore are not restricted to homopurine targets.
Pseudocomplementary oligonucleotides can be designed for mixed,
general sequence recognition of a desired target site. Preferably,
the target site contains an A:T base pair content of about 40% or
greater. Preferably pseudocomplementary oligonucleotides are
between about 8 and 50 nucleobases, more preferably 8 to 30, even
more preferably between about 8 and 20 nucleobases.
The pseudocomplementary oligonucleotides should be designed to bind
to the target site (DDTS) at a distance of between about 1 to 800
bases from the target site of the donor oligonucleotide. More
preferably, the pseudocomplementary oligonucleotides bind at a
distance of between about 25 and 75 bases from the donor
oligonucleotide. Most preferably, the pseudocomplementary
oligonucleotides bind at a distance of about 50 bases from the
donor oligonucleotide. Preferred pcPNA sequences for targeted
repair of a mutation in the .beta.-globin intron IVS2 (G to A) are
described in U.S. Pat. No. 8,309,356.
Preferably, the pseudocomplementary oligonucleotides bind/hybridize
to the target nucleic acid molecule under conditions of high
stringency and specificity. Most preferably, the oligonucleotides
bind in a sequence-specific manner and induce the formation of
double duplex. Specificity and binding affinity of the
pseudocomplementary oligonucleotides may vary from oligonucleotide
to oligonucleotide, depending on factors such as oligonucleotide
length, the number of G:C and A:T base pairs, and the
formulation.
C. CRISPR/Cas
In some embodiments, the gene editing composition is the CRISPR/Cas
system. CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) is an acronym for DNA loci that contain multiple, short,
direct repetitions of base sequences. The prokaryotic CRISPR/Cas
system has been adapted for use as gene editing (silencing,
enhancing or changing specific genes) for use in eukaryotes (see,
for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek,
et al., Science, 337(6096):816-21 (2012)). By transfecting a cell
with the required elements including a cas gene and specifically
designed CRISPRs, the organism's genome can be cut and modified at
any desired location. Methods of preparing compositions for use in
genome editing using the CRISPR/Cas systems are described in detail
in WO 2013/176772 and WO 2014/018423, which are specifically
incorporated by reference herein in their entireties.
In general, "CRISPR system" refers collectively to transcripts and
other elements involved in the expression of or directing the
activity of CRISPR-associated ("Cas") genes, including sequences
encoding a Cas gene, a tracr (trans-activating CRISPR) sequence
(e.g., tracrRNA or an active partial tracrRNA), a tracr-mate
sequence (encompassing a "direct repeat" and a tracrRNA-processed
partial direct repeat in the context of an endogenous CRISPR
system), a guide sequence (also referred to as a "spacer" in the
context of an endogenous CRISPR system), or other sequences and
transcripts from a CRISPR locus. One or more tracr mate sequences
operably linked to a guide sequence (e.g., direct
repeat-spacer-direct repeat) can also be referred to as pre-crRNA
(pre-CRISPR RNA) before processing or crRNA after processing by a
nuclease.
In some embodiments, a tracrRNA and crRNA are linked and form a
chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a
partial tracrRNA via a synthetic stem loop to mimic the natural
crRNA:tracrRNA duplex as described in Cong, Science,
15:339(6121):819-823 (2013) and Jinek, et al., Science,
337(6096):816-21 (2012)). A single fused crRNA-tracrRNA construct
can also be referred to as a guide RNA or gRNA (or single-guide RNA
(sgRNA)). Within an sgRNA, the crRNA portion can be identified as
the "target sequence" and the tracrRNA is often referred to as the
"scaffold."
There are many resources available for helping practitioners
determine suitable target sites once a desired DNA target sequence
is identified. For example, numerous public resources, including a
bioinformatically generated list of about 190,000 potential sgRNAs,
targeting more than 40% of human exons, are available to aid
practitioners in selecting target sites and designing the associate
sgRNA to affect a nick or double strand break at the site. See
also, crispr.u-psud.fr/, a tool designed to help scientists find
CRISPR targeting sites in a wide range of species and generate the
appropriate crRNA sequences.
In some embodiments, one or more vectors driving expression of one
or more elements of a CRISPR system are introduced into a target
cell such that expression of the elements of the CRISPR system
direct formation of a CRISPR complex at one or more target sites.
While the specifics can be varied in different engineered CRISPR
systems, the overall methodology is similar. A practitioner
interested in using CRISPR technology to target a DNA sequence
(such as CTPS1) can insert a short DNA fragment containing the
target sequence into a guide RNA expression plasmid. The sgRNA
expression plasmid contains the target sequence (about 20
nucleotides), a form of the tracrRNA sequence (the scaffold) as
well as a suitable promoter and necessary elements for proper
processing in eukaryotic cells. Such vectors are commercially
available (see, for example, Addgene). Many of the systems rely on
custom, complementary oligos that are annealed to form a double
stranded DNA and then cloned into the sgRNA expression plasmid.
Co-expression of the sgRNA and the appropriate Cas enzyme from the
same or separate plasmids in transfected cells results in a single
or double strand break (depending of the activity of the Cas
enzyme) at the desired target site.
D. Zinc Finger Nucleases
In some embodiments, the element that induces a single or a double
strand break in the target cell's genome is a nucleic acid
construct or constructs encoding a zinc finger nucleases (ZFNs).
ZFNs are typically fusion proteins that include a DNA-binding
domain derived from a zinc-finger protein linked to a cleavage
domain.
The most common cleavage domain is the Type IIS enzyme Fok1. Fok1
catalyzes double-stranded cleavage of DNA, at 9 nucleotides from
its recognition site on one strand and .beta. nucleotides from its
recognition site on the other. See, for example, U.S. Pat. Nos.
5,356,802; 5,436,150 and 5,487,994; as well as Li et al. Proc.,
Natl. Acad. Sci. USA 89 (1992):4275-4279; Li et al. Proc. Natl.
Acad. Sci. USA, 90:2764-2768 (1993); Kim et al. Proc. Natl. Acad.
Sci. USA. 91:883-887 (1994a); Kim et al. J. Biol. Chem.
269:31,978-31,982 (1994b). One or more of these enzymes (or
enzymatically functional fragments thereof) can be used as a source
of cleavage domains.
The DNA-binding domain, which can, in principle, be designed to
target any genomic location of interest, can be a tandem array of
Cys.sub.2His.sub.2 zinc fingers, each of which generally recognizes
three to four nucleotides in the target DNA sequence. The
Cys.sub.2His.sub.2 domain has a general structure: Phe (sometimes
Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)-Phe(sometimes
Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His.
By linking together multiple fingers (the number varies: three to
six fingers have been used per monomer in published studies), ZFN
pairs can be designed to bind to genomic sequences 18-36
nucleotides long.
Engineering methods include, but are not limited to, rational
design and various types of empirical selection methods. Rational
design includes, for example, using databases including triplet (or
quadruplet) nucleotide sequences and individual zinc finger amino
acid sequences, in which each triplet or quadruplet nucleotide
sequence is associated with one or more amino acid sequences of
zinc fingers which bind the particular triplet or quadruplet
sequence. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242;
6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S.
Published Application Nos. 2002/0165356; 2004/0197892;
2007/0154989; 2007/0213269; and International Patent Application
Publication Nos. WO 98/53059 and WO 2003/016496.
E. Transcription Activator-Like Effector Nucleases
In some embodiments, the element that induces a single or a double
strand break in the target cell's genome is a nucleic acid
construct or constructs encoding a transcription activator-like
effector nuclease (TALEN). TALENs have an overall architecture
similar to that of ZFNs, with the main difference that the
DNA-binding domain comes from TAL effector proteins, transcription
factors from plant pathogenic bacteria. The DNA-binding domain of a
TALEN is a tandem array of amino acid repeats, each about 34
residues long. The repeats are very similar to each other;
typically they differ principally at two positions (amino acids 12
and 13, called the repeat variable diresidue, or RVD). Each RVD
specifies preferential binding to one of the four possible
nucleotides, meaning that each TALEN repeat binds to a single base
pair, though the NN RVD is known to bind adenines in addition to
guanine. TAL effector DNA binding is mechanistically less well
understood than that of zinc-finger proteins, but their seemingly
simpler code could prove very beneficial for engineered-nuclease
design. TALENs also cleave as dimers, have relatively long target
sequences (the shortest reported so far binds 13 nucleotides per
monomer) and appear to have less stringent requirements than ZFNs
for the length of the spacer between binding sites. Monomeric and
dimeric TALENs can include more than 10, more than 14, more than
20, or more than 24 repeats.
Methods of engineering TAL to bind to specific nucleic acids are
described in Cermak, et al, Nucl. Acids Res. 1-11 (2011). U.S.
Published Application No. 2011/0145940, which discloses TAL
effectors and methods of using them to modify DNA. Miller et al.
Nature Biotechnol 29: 143 (2011) reported making TALENs for
site-specific nuclease architecture by linking TAL truncation
variants to the catalytic domain of Fok1 nuclease. The resulting
TALENs were shown to induce gene modification in immortalized human
cells. General design principles for TALE binding domains can be
found in, for example, WO 2011/072246.
IV. Donor Oligonucleotides
In some embodiments, the gene editing composition includes or is
administered in combination with a donor oligonucleotide.
Generally, in the case of gene therapy, the donor oligonucleotide
includes a sequence that can correct a mutation(s) in the host
genome, though in some embodiments, the donor introduces a mutation
that can, for example, reduce expression of an oncogene or a
receptor that facilitates HIV infection. In addition to containing
a sequence designed to introduce the desired correction or
mutation, the donor oligonucleotide may also contain synonymous
(silent) mutations (e.g., 7 to 10). The additional silent mutations
can facilitate detection of the corrected target sequence using
allele-specific PCR of genomic DNA isolated from treated cells.
A. Preferred Donor Oligonucleotide Design for Triplex and
Double-Duplex based Technologies
The triplex forming molecules including peptide nucleic acids may
be administered in combination with, or tethered to, a donor
oligonucleotide via a mixed sequence linker or used in conjunction
with a non-tethered donor oligonucleotide that is substantially
homologous to the target sequence. Triplex-forming molecules can
induce recombination of a donor oligonucleotide sequence up to
several hundred base pairs away. It is preferred that the donor
oligonucleotide sequence is between 1 to 800 bases from the target
binding site of the triplex-forming molecules. More preferably the
donor oligonucleotide sequence is between 25 to 75 bases from the
target binding site of the triplex-forming molecules. Most
preferably that the donor oligonucleotide sequence is about 50
nucleotides from the target binding site of the triplex-forming
molecules.
The donor sequence can contain one or more nucleic acid sequence
alterations compared to the sequence of the region targeted for
recombination, for example, a substitution, a deletion, or an
insertion of one or more nucleotides. Successful recombination of
the donor sequence results in a change of the sequence of the
target region. Donor oligonucleotides are also referred to herein
as donor fragments, donor nucleic acids, donor DNA, or donor DNA
fragments. This strategy exploits the ability of a triplex to
provoke DNA repair, potentially increasing the probability of
recombination with the homologous donor DNA. It is understood in
the art that a greater number of homologous positions within the
donor fragment will increase the probability that the donor
fragment will be recombined into the target sequence, target
region, or target site. Tethering of a donor oligonucleotide to a
triplex-forming molecule facilitates target site recognition via
triple helix formation while at the same time positioning the
tethered donor fragment for possible recombination and information
transfer. Triplex-forming molecules also effectively induce
homologous recombination of non-tethered donor oligonucleotides.
The term "recombinagenic" as used herein, is used to define a DNA
fragment, oligonucleotide, peptide nucleic acid, or composition as
being able to recombine into a target site or sequence or induce
recombination of another DNA fragment, oligonucleotide, or
composition.
Non-tethered or unlinked fragments may range in length from 20
nucleotides to several thousand. The donor oligonucleotide
molecules, whether linked or unlinked, can exist in single stranded
or double stranded form. The donor fragment to be recombined can be
linked or un-linked to the triplex forming molecules. The linked
donor fragment may range in length from 4 nucleotides to 100
nucleotides, preferably from 4 to 80 nucleotides in length.
However, the unlinked donor fragments have a much broader range,
from 20 nucleotides to several thousand. In one embodiment the
oligonucleotide donor is between 25 and 80 nucleobases. In a
further embodiment, the non-tethered donor nucleotide is about 50
to 60 nucleotides in length.
The donor oligonucleotides contain at least one mutated, inserted
or deleted nucleotide relative to the target DNA sequence. Target
sequences can be within the coding DNA sequence of the gene or
within introns. Target sequences can also be within DNA sequences
which regulate expression of the target gene, including promoter or
enhancer sequences or sequences that regulate RNA splicing.
The donor oligonucleotides can contain a variety of mutations
relative to the target sequence. Representative types of mutations
include, but are not limited to, point mutations, deletions and
insertions. Deletions and insertions can result in frameshift
mutations or deletions. Point mutations can cause missense or
nonsense mutations. These mutations may disrupt, reduce, stop,
increase, improve, or otherwise alter the expression of the target
gene.
Compositions including triplex-forming molecules such as tcPNA may
include one or more than one donor oligonucleotides. More than one
donor oligonucleotides may be administered with triplex-forming
molecules in a single transfection, or sequential transfections.
Use of more than one donor oligonucleotide may be useful, for
example, to create a heterozygous target gene where the two alleles
contain different modifications.
Donor oligonucleotides are preferably DNA oligonucleotides,
composed of the principal naturally-occurring nucleotides (uracil,
thymine, cytosine, adenine and guanine) as the heterocyclic bases,
deoxyribose as the sugar moiety, and phosphate ester linkages.
Donor oligonucleotides may include modifications to nucleobases,
sugar moieties, or backbone/linkages, as described above, depending
on the desired structure of the replacement sequence at the site of
recombination or to provide some resistance to degradation by
nucleases. Modifications to the donor oligonucleotide should not
prevent the donor oligonucleotide from successfully recombining at
the recombination target sequence in the presence of
triplex-forming molecules.
B. Preferred Donor Oligonucleotides Design for Nuclease-based
Technologies
The nuclease activity of the genome editing systems described
herein cleave target DNA to produce single or double strand breaks
in the target DNA. Double strand breaks can be repaired by the cell
in one of two ways: non-homologous end joining, and
homology-directed repair. In non-homologous end joining (NHEJ), the
double-strand breaks are repaired by direct ligation of the break
ends to one another. As such, no new nucleic acid material is
inserted into the site, although some nucleic acid material may be
lost, resulting in a deletion. In homology-directed repair, a donor
polynucleotide with homology to the cleaved target DNA sequence is
used as a template for repair of the cleaved target DNA sequence,
resulting in the transfer of genetic information from a donor
polynucleotide to the target DNA. As such, new nucleic acid
material can be inserted/copied into the site.
Therefore, in some embodiments, the genome editing composition
optionally includes a donor polynucleotide. The modifications of
the target DNA due to NHEJ and/or homology-directed repair can be
used to induce gene correction, gene replacement, gene tagging,
transgene insertion, nucleotide deletion, gene disruption, gene
mutation, etc.
Accordingly, cleavage of DNA by the genome editing composition can
be used to delete nucleic acid material from a target DNA sequence
by cleaving the target DNA sequence and allowing the cell to repair
the sequence in the absence of an exogenously provided donor
polynucleotide. Alternatively, if the genome editing composition
includes a donor polynucleotide sequence that includes at least a
segment with homology to the target DNA sequence, the methods can
be used to add, i.e., insert or replace, nucleic acid material to a
target DNA sequence (e.g., to "knock in" a nucleic acid that
encodes for a protein, an siRNA, an miRNA, etc.), to add a tag
(e.g., 6.times.His, a fluorescent protein (e.g., a green
fluorescent protein; a yellow fluorescent protein, etc.),
hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a
gene (e.g., promoter, polyadenylation signal, internal ribosome
entry sequence (IRES), 2A peptide, start codon, stop codon, splice
signal, localization signal, etc.), to modify a nucleic acid
sequence (e.g., introduce a mutation), and the like. As such, the
compositions can be used to modify DNA in a site-specific, i.e.,
"targeted", way, for example gene knock-out, gene knock-in, gene
editing, gene tagging, etc. as used in, for example, gene
therapy.
In applications in which it is desirable to insert a polynucleotide
sequence into a target DNA sequence, a polynucleotide including a
donor sequence to be inserted is also provided to the cell. By a
"donor sequence" or "donor polynucleotide" or "donor
oligonucleotide" it is meant a nucleic acid sequence to be inserted
at the cleavage site. The donor polynucleotide typically contains
sufficient homology to a genomic sequence at the cleavage site,
e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide
sequences flanking the cleavage site, e.g., within about 50 bases
or less of the cleavage site, e.g., within about 30 bases, within
about 15 bases, within about 10 bases, within about 5 bases, or
immediately flanking the cleavage site, to support
homology-directed repair between it and the genomic sequence to
which it bears homology. The donor sequence is typically not
identical to the genomic sequence that it replaces. Rather, the
donor sequence may contain at least one or more single base
changes, insertions, deletions, inversions or rearrangements with
respect to the genomic sequence, so long as sufficient homology is
present to support homology-directed repair. In some embodiments,
the donor sequence includes a non-homologous sequence flanked by
two regions of homology, such that homology-directed repair between
the target DNA region and the two flanking sequences results in
insertion of the non-homologous sequence at the target region.
V. Oligonucleotide Composition
Any of the gene editing technologies, components thereof, donor
oligonucleotides, or other nucleic acids disclosed herein can
include one or more modifications or substitutions to the
nucleobases or linkages. Although modifications are particularly
preferred for use with triplex-forming technologies and typically
discussed below with reference thereto, any of the modifications
can be utilized in the construction of any of the disclosed gene
editing compositions, donor, nucleotides, etc. Modifications should
not prevent, and preferably enhance the activity, persistence, or
function of the gene editing technology. For example, modifications
to oligonucleotides for use as triplex-forming should not prevent,
and preferably enhance duplex invasion, strand displacement, and/or
stabilize triplex formation as described above by increasing
specificity or binding affinity of the triplex-forming molecules to
the target site. Modified bases and base analogues, modified sugars
and sugar analogues and/or various suitable linkages known in the
art are also suitable for use in the molecules disclosed herein.
Several preferred oligonucleotide compositions including PNA, and
modification thereof to include MiniPEG at the .gamma. position in
the PNA backbone, are discussed above. Additional modifications are
discussed in more detail below.
A. Heterocyclic Bases
The principal naturally-occurring nucleotides include uracil,
thymine, cytosine, adenine and guanine as the heterocyclic bases.
Gene editing molecules can include chemical modifications to their
nucleotide constituents. For example, target sequences with
adjacent cytosines can be problematic. Triplex stability is greatly
compromised by runs of cytosines, thought to be due to repulsion
between the positive charge resulting from the N.sup.3 protonation
or perhaps because of competition for protons by the adjacent
cytosines. Chemical modification of nucleotides including
triplex-forming molecules such as PNAs may be useful to increase
binding affinity of triplex-forming molecules and/or triplex
stability under physiologic conditions.
Chemical modifications of heterocyclic bases or heterocyclic base
analogs may be effective to increase the binding affinity of a
nucleotide or its stability in a triplex. Chemically-modified
heterocyclic bases include, but are not limited to, inosine,
5-(1-propynyl) uracil (pU), 5-(1-propynyl) cytosine (pC),
5-methylcytosine, 8-oxo-adenine, pseudocytosine, pseudoisocytosine,
5 and 2-amino-5-(2'-deoxy-.beta.-D-ribofuranosyl)pyridine
(2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine
derivatives. Substitution of 5-methylcytosine or pseudoisocytosine
for cytosine in triplex-forming molecules such as PNAs helps to
stabilize triplex formation at neutral and/or physiological pH,
especially in triplex-forming molecules with isolated cytosines.
This is because the positive charge partially reduces the negative
charge repulsion between the triplex-forming molecules and the
target duplex, and allows for Hoogsteen binding.
B. Backbone
The nucleotide subunits of the triplex-forming molecules such as
PNAs are connected by an internucleotide bond that refers to a
chemical linkage between two nucleoside moieties. Peptide nucleic
acids (PNAs) are synthetic DNA mimics in which the phosphate
backbone of the oligonucleotide is replaced in its entirety by
repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds
are typically replaced by peptide bonds. The various heterocyclic
bases are linked to the backbone by methylene carbonyl bonds, which
allow them to form PNA-DNA or PNA-RNA duplexes via Watson-Crick
base pairing with high affinity and sequence-specificity. PNAs
maintain spacing of heterocyclic bases that is similar to
conventional DNA oligonucleotides, but are achiral and neutrally
charged molecules. Peptide nucleic acids are composed of peptide
nucleic acid monomers.
Other backbone modifications, particularly those relating to PNAs,
include peptide and amino acid variations and modifications. Thus,
the backbone constituents of PNAs may be peptide linkages, or
alternatively, they may be non-peptide linkages. Examples include
acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid
(referred to herein as O-linkers), amino acids such as lysine are
particularly useful if positive charges are desired in the PNA, and
the like. Methods for the chemical assembly of PNAs are well known.
See, for example, U.S. Pat. Nos. 5,539,082, 5,527,675, 5,623,049,
5,714,331, 5,736,336, 5,773,571 and 5,786,571.
Backbone modifications used to generate triplex-forming molecules
should not prevent the molecules from binding with high specificity
to the target site and creating a triplex with the target duplex
nucleic acid by displacing one strand of the target duplex and
foiming a clamp around the other strand of the target duplex.
C. Modified Nucleic Acids
Modified nucleic acids in addition to peptide nucleic acids are
also useful as triplex-forming molecules. Oligonucleotides are
composed a chain of nucleotides which are linked to one another.
Canonical nucleotides typically include a heterocyclic base
(nucleic acid base), a sugar moiety attached to the heterocyclic
base, and a phosphate moiety which esterifies a hydroxyl function
of the sugar moiety. The principal naturally-occurring nucleotides
include uracil, thymine, cytosine, adenine and guanine as the
heterocyclic bases, and ribose or deoxyribose sugar linked by
phosphodiester bonds. As used herein "modified nucleotide" or
"chemically modified nucleotide" defines a nucleotide that has a
chemical modification of one or more of the heterocyclic base,
sugar moiety or phosphate moiety constituents. Preferably the
charge of the modified nucleotide is reduced compared to DNA or RNA
oligonucleotides of the same nucleobase sequence. Most preferably
the triplex-forming molecules have low negative charge, no charge,
or positive charge such that electrostatic repulsion with the
nucleotide duplex at the target site is reduced compared to DNA or
RNA oligonucleotides with the corresponding nucleobase
sequence.
Examples of modified nucleotides with reduced charge include
modified internucleotide linkages such as phosphate analogs having
achiral and uncharged intersubunit linkages (e.g., Sterchak, E. P.
et al., Organic Chem., 52:4202, (1987)), and uncharged
morpholino-based polymers having achiral intersubunit linkages
(see, e.g., U.S. Pat. No. 5,034,506). Some internucleotide linkage
analogs include morpholidate, acetal, and polyamide-linked
heterocycles. Locked nucleic acids (LNA) are modified RNA
nucleotides (see, for example, Braasch, et al., Chem. Biol.,
8(1):1-7 (2001)). LNAs form hybrids with DNA which are more stable
than DNA/DNA hybrids, a property similar to that of peptide nucleic
acid (PNA)/DNA hybrids. Therefore, LNA can be used just as PNA
molecules would be. LNA binding efficiency can be increased in some
embodiments by adding positive charges to it. Commercial nucleic
acid synthesizers and standard phosphoramidite chemistry are used
to make LNAs.
Molecules may also include nucleotides with modified heterocyclic
bases, sugar moieties or sugar moiety analogs. Modified nucleotides
may include modified heterocyclic bases or base analogs as
described above with respect to peptide nucleic acids. Sugar moiety
modifications include, but are not limited to, 2'-O-aminoethoxy,
2'-O-amonioethyl (2'-OAE), 2'-O-methoxy, 2'-O-methyl,
2-guanidoethyl (2'-OGE), 2'-O,4'-C-methylene (LNA),
2'-O-(methoxyethyl) (2'-OME) and 2'-O--(N-(methyl)acetamido)
(2'-OMA). 2'-O-aminoethyl sugar moiety substitutions are especially
preferred because they are protonated at neutral pH and thus
suppress the charge repulsion between the triplex-forming molecule
and the target duplex. This modification stabilizes the C3'-endo
conformation of the ribose or deoxyribose and also forms a bridge
with the i-1 phosphate in the purine strand of the duplex.
VI. Nanoparticle Delivery Vehicles
Any of the disclosed compositions including, but not limited to
potentiating factors, gene editing molecules, donor
oligonucleotides, etc., can be delivered to the target cells using
a nanoparticle delivery vehicle. In some embodiments, some of the
compositions are packaged in nanoparticles and some are not. For
example, in some embodiments, the gene editing technology and/or
donor oligonucleotide is incorporated into nanoparticles while the
potentiating factor is not. In some embodiments, the gene editing
technology and/or donor oligonucleotide, and the potentiating
factor are packaged in nanoparticles. The different compositions
can be packaged in the same nanoparticles or different
nanoparticles. For example, the compositions can be mixed and
packaged together. In some embodiments, the different compositions
are packaged separately into separate nanoparticles wherein the
nanoparticles are similarly or identically composed and/or
manufactured. In some embodiments, the different compositions are
packaged separately into separate nanoparticles wherein the
nanoparticles are differentially composed and/or manufactured.
Nanoparticles generally refers to particles in the range of between
500 nm to less than 0.5 nm, preferably having a diameter that is
between 50 and 500 nm, more preferably having a diameter that is
between 50 and 300 nm. Cellular internalization of polymeric
particles is highly dependent upon their size, with nanoparticulate
polymeric particles being internalized by cells with much higher
efficiency than microparticulate polymeric particles. For example,
Desai, et al. have demonstrated that about 2.5 times more
nanoparticles that are 100 nm in diameter are taken up by cultured
Caco-2 cells as compared to microparticles having a diameter on 1
.mu.M (Desai, et al., Pharm. Res., 14:1568-73 (1997)).
Nanoparticles also have a greater ability to diffuse deeper into
tissues in vivo.
A. Polymer
The polymer that forms the core of the nanoparticle may be any
biodegradable or non-biodegradable synthetic or natural polymer. In
a preferred embodiment, the polymer is a biodegradable polymer.
Nanoparticles are ideal materials for the fabrication of gene
editing delivery vehicles: 1) control over the size range of
fabrication, down to 100 nm or less, an important feature for
passing through biological barriers; 2) reproducible
biodegradability without the addition of enzymes or cofactors; 3)
capability for sustained release of encapsulated, protected nucleic
acids over a period in the range of days to months by varying
factors such as the monomer ratios or polymer size, for example,
the ratio of lactide to glycolide monomer units in
poly(lactide-co-glycolide) (PLGA); 4) well-understood fabrication
methodologies that offer flexibility over the range of parameters
that can be used for fabrication, including choices of the polymer
material, solvent, stabilizer, and scale of production; and 5)
control over surface properties facilitating the introduction of
modular functionalities into the surface.
Examples of preferred biodegradable polymers include synthetic
polymers that degrade by hydrolysis such as poly(hydroxy acids),
such as polymers and copolymers of lactic acid and glycolic acid,
other degradable polyesters, polyanhydrides, poly(ortho)esters,
polyesters, polyurethanes, poly(butic acid), poly(valeric acid),
poly(caprolactone), poly(hydroxyalkanoates),
poly(lactide-co-caprolactone), and poly(amine-co-ester) polymers,
such as those described in Zhou, et al., Nature Materials, 11:82-90
(2012) and WO 2013/082529, U.S. Published Application No.
2014/0342003, and PCT/US2015/061375.
Preferred natural polymers include alginate and other
polysaccharides, collagen, albumin and other hydrophilic proteins,
zein and other prolamines and hydrophobic proteins, copolymers and
mixtures thereof. In general, these materials degrade either by
enzymatic hydrolysis or exposure to water in vivo, by surface or
bulk erosion.
In some embodiments, non-biodegradable polymers can be used,
especially hydrophobic polymers. Examples of preferred
non-biodegradable polymers include ethylene vinyl acetate,
poly(meth) acrylic acid, copolymers of maleic anhydride with other
unsaturated polymerizable monomers, poly(butadiene maleic
anhydride), polyamides, copolymers and mixtures thereof, and
dextran, cellulose and derivatives thereof.
Other suitable biodegradable and non-biodegradable polymers
include, but are not limited to, polyanhydrides, polyamides,
polycarbonates, polyalkylenes, polyalkylene oxides such as
polyethylene glycol, polyalkylene terepthalates such as
poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers,
polyvinyl esters, polyethylene, polypropylene, poly(vinyl acetate),
poly vinyl chloride, polystyrene, polyvinyl halides,
polyvinylpyrrolidone, polymers of acrylic and methacrylic esters,
polysiloxanes, polyurethanes and copolymers thereof, modified
celluloses, alkyl cellulose, hydroxyalkyl celluloses, cellulose
ethers, cellulose esters, nitro celluloses, cellulose acetate,
cellulose propionate, cellulose acetate butyrate, cellulose acetate
phthalate, carboxyethyl cellulose, cellulose triacetate, cellulose
sulfate sodium salt, and polyacrylates such as poly(methyl
methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate),
poly(isobutylmethacrylate), poly(hexylmethacrylate),
poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate). These materials
may be used alone, as physical mixtures (blends), or as
co-polymers.
The polymer may be a bioadhesive polymer that is hydrophilic or
hydrophobic. Hydrophilic polymers include CARBOPOL.TM. (a high
molecular weight, crosslinked, acrylic acid-based polymers
manufactured by NOVEON.TM.), polycarbophil, cellulose esters, and
dextran.
Release rate controlling polymers may be included in the polymer
matrix or in the coating on the formulation. Examples of rate
controlling polymers that may be used are
hydroxypropylmethylcellulose (HPMC) with viscosities of either 5,
50, 100 or 4000 cps or blends of the different viscosities,
ethylcellulose, methylmethacrylates, such as EUDRAGIT.RTM. RS100,
EUDRAGIT.RTM. RL100, EUDRAGIT.RTM. NE 30D (supplied by Rohm
America). Gastrosoluble polymers, such as EUDRAGIT.RTM. E100 or
enteric polymers such as EUDRAGIT.RTM. L100-55D, L100 and 5100 may
be blended with rate controlling polymers to achieve pH dependent
release kinetics. Other hydrophilic polymers such as alginate,
polyethylene oxide, carboxymethylcellulose, and
hydroxyethylcellulose may be used as rate controlling polymers.
These polymers can be obtained from sources such as Sigma Chemical
Co., St. Louis, Mo.; Polysciences, Warrenton, Pa.; Aldrich,
Milwaukee, Wis.; Fluka, Ronkonkoma, N.Y.; and BioRad, Richmond,
Calif., or can be synthesized from monomers obtained from these or
other suppliers using standard techniques.
In a preferred embodiment, the nanoparticles are formed of polymers
fabricated from polylactides (PLA) and copolymers of lactide and
glycolide (PLGA). These have established commercial use in humans
and have a long safety record (Jiang, et al., Adv. Drug Deliv.
Rev., 57(3):391-410); Aguado and Lambert, Immunobiology,
184(2-3):113-25 (1992); Bramwell, et al., Adv. Drug Deliv. Rev.,
57(9):1247-65 (2005)). These polymers have been used to encapsulate
siRNA (Yuan, et al., Jour. Nanosocience and Nanotechnology,
6:2821-8 (2006); Braden, et al., Jour. Biomed. Nanotechnology,
3:148-59 (2007); Khan, et al., Jour. Drug Target, 12:393-404
(2004); Woodrow, et al., Nature Materials, 8:526-533 (2009)).
Murata, et al., J. Control. Release, 126(3):246-54 (2008) showed
inhibition of tumor growth after intratumoral injection of PLGA
microspheres encapsulating siRNA targeted against vascular
endothelial growth factor (VEGF). However, these microspheres were
too large to be endocytosed (35-45 .mu.m) (Conner and Schmid,
Nature, 422(6927):37-44 (2003)) and required release of the
anti-VEGF siRNA extracellularly as a polyplex with either
polyarginine or PEI before they could be internalized by the cell.
These microparticles may have limited applications because of the
toxicity of the polycations and the size of the particles.
Nanoparticles (100-300 nm) of PLGA can penetrate deep into tissue
and are easily internalized by many cells (Conner and Schmid,
Nature, 422(6927):37-44 (2003)).
The nanoparticles can be designed to release encapsulated nucleic
acids over a period of days to weeks. Factors that affect the
duration of release include pH of the surrounding medium (higher
rate of release at pH 5 and below due to acid catalyzed hydrolysis
of PLGA) and polymer composition. Aliphatic polyesters differ in
hydrophobicity, affecting degradation rate. Specifically, the
hydrophobic poly (lactic acid) (PLA), more hydrophilic poly
(glycolic acid) PGA and their copolymers, poly
(lactide-co-glycolide) (PLGA) have various release rates. The
degradation rate of these polymers, and often the corresponding
drug release rate, can vary from days (PGA) to months (PLA) and is
easily manipulated by varying the ratio of PLA to PGA.
Exemplary nanoparticles are described in U.S. Pat. Nos. 4,883,666,
5,114,719, 5,601,835, 7,534,448, 7,534,449, 7,550,154, and
8,889,117, and U.S. Published Application Nos. 2009/0269397,
2009/0239789, 2010/0151436, 2011/0008451, 2011/0268810,
2014/0342003, 2015/0118311, 2015/0125384, 2015/0073041, Hubbell, et
al., Science, 337:303-305 (2012), Cheng, et al., Biomaterials,
32:6194-6203 (2011), Rodriguez, et al., Science, 339:971-975
(2013), Hrkach, et al., Sci Transl Med., 4:128ra139 (2012), McNeer,
et al., Mol Ther., 19:172-180 (2011), McNeer, et al., Gene Ther.,
20:658-659 (2013), Babar, et al., Proc Natl Acad Sci USA,
109:E1695-E1704 (2012), Fields, et al., J Control Release 164:41-48
(2012), and Fields, et al., Advanced Healthcare Materials, 361-366
(2015).
B. Polycations
In a preferred embodiment, the nucleic acids are complexed to
polycations to increase the encapsulation efficiency of the nucleic
acids into the nanoparticles. The term "polycation" refers to a
compound having a positive charge, preferably at least 2 positive
charges, at a selected pH, preferably physiological pH.
Polycationic moieties have between about 2 to about 15 positive
charges, preferably between about 2 to about 12 positive charges,
and more preferably between about 2 to about 8 positive charges at
selected pH values.
Many polycations are known in the art. Suitable constituents of
polycations include basic amino acids and their derivatives such as
arginine, asparagine, glutamine, lysine and histidine; cationic
dendrimers; and amino polysaccharides. Suitable polycations can be
linear, such as linear tetralysine, branched or dendrimeric in
structure.
Exemplary polycations include, but are not limited to, synthetic
polycations based on acrylamide and
2-acrylamido-2-methylpropanetrimethylamine,
poly(N-ethyl-4-vinylpyridine) or similar quartemized polypyridine,
diethylaminoethyl polymers and dextran conjugates, polymyxin B
sulfate, lipopolyamines, poly(allylamines) such as the strong
polycation poly(dimethyldiallylammonium chloride),
polyethyleneimine, polybrene, and polypeptides such as protamine,
the histone polypeptides, polylysine, polyarginine and
polyornithine.
In one embodiment, the polycation is a polyamine. Polyamines are
compounds having two or more primary amine groups. In a preferred
embodiment, the polyamine is a naturally occurring polyamine that
is produced in prokaryotic or eukaryotic cells. Naturally occurring
polyamines represent compounds with cations that are found at
regularly-spaced intervals and are therefore particularly suitable
for complexing with nucleic acids. Polyamines play a major role in
very basic genetic processes such as DNA synthesis and gene
expression. Polyamines are integral to cell migration,
proliferation and differentiation in plants and animals. The
metabolic levels of polyamines and amino acid precursors are
critical and hence biosynthesis and degradation are tightly
regulated. Suitable naturally occurring polyamines include, but are
not limited to, spermine, spermidine, cadaverine and putrescine. In
a preferred embodiment, the polyamine is spermidine.
In another embodiment, the polycation is a cyclic polyamine. Cyclic
polyamines are known in the art and are described, for example, in
U.S. Pat. No. 5,698,546, WO 1993/012096 and WO 2002/010142.
Exemplary cyclic polyamines include, but are not limited to,
cyclen.
Spermine and spermidine are derivatives of putrescine
(1,4-diaminobutane) which is produced from L-omithine by action of
ODC (ornithine decarboxylase). L-ornithine is the product of
L-arginine degradation by arginase. Spermidine is a triamine
structure that is produced by spermidine synthase (SpdS) which
catalyzes monoalkylation of putrescine (1,4-diaminobutane) with
decarboxylated S-adenosylmethionine (dcAdoMet) 3-aminopropyl donor.
The formal alkylation of both amino groups of putrescine with the
3-aminopropyl donor yields the symmetrical tetraamine spermine. The
biosynthesis of spermine proceeds to spermidine by the effect of
spermine synthase (SpmS) in the presence of dcAdoMet. The
3-aminopropyl donor (dcAdoMet) is derived from S-adenosylmethionine
by sequential transformation of L-methionine by methionine
adenosyltransferase followed by decarboxylation by AdoMetDC
(S-adenosylmethionine decarboxylase). Hence, putrescine, spermidine
and spermine are metabolites derived from the amino acids
L-arginine (L-ornithine, putrescine) and L-methionine (dcAdoMet,
aminopropyl donor).
In some embodiments, the particles themselves are a polycation
(e.g., a blend of PLGA and poly(beta amino ester).
C. Coupling Agents or Ligands
The external surface of the polymeric nanoparticles may be modified
by conjugating to, or incorporating into, the surface of the
nanoparticle a coupling agent or ligand.
In a preferred embodiment, the coupling agent is present in high
density on the surface of the nanoparticle. As used herein, "high
density" refers to polymeric nanoparticles having a high density of
ligands or coupling agents, which is preferably in the range of
1,000 to 10,000,000, more preferably 10,000-1,000,000 ligands per
square micron of nanoparticle surface area. This can be measured by
fluorescence staining of dissolved particles and calibrating this
fluorescence to a known amount of free fluorescent molecules in
solution.
Coupling agents associate with the polymeric nanoparticles and
provide substrates that facilitate the modular assembly and
disassembly of functional elements to the nanoparticles. Coupling
agents or ligands may associate with nanoparticles through a
variety of interactions including, but not limited to, hydrophobic
interactions, electrostatic interactions and covalent coupling.
In a preferred embodiment, the coupling agents are molecules that
match the polymer phase hydrophile-lipophile balance.
Hydrophile-lipophile balances range from 1 to 15. Molecules with a
low hydrophile-lipophile balance are more lipid loving and thus
tend to make a water in oil emulsion while those with a high
hydrophile-lipophile balance are more hydrophilic and tend to make
an oil in water emulsion. Fatty acids and lipids have a low
hydrophile-lipophile balance below 10.
Any amphiphilic polymer with a hydrophile-lipophile balance in the
range 1-10, more preferably between 1 and 6, most preferably
between 1 and up to 5, can be used as a coupling agent. Examples of
coupling agents which may associate with polymeric nanoparticles
via hydrophobic interactions include, but are not limited to, fatty
acids, hydrophobic or amphipathic peptides or proteins, and
polymers. These classes of coupling agents may also be used in any
combination or ratio. In a preferred embodiment, the association of
adaptor elements with nanoparticles facilitates a prolonged
presentation of functional elements which can last for several
weeks.
Coupling agents can also be attached to polymeric nanoparticles
through covalent interactions through various functional groups.
Functionality refers to conjugation of a molecule to the surface of
the particle via a functional chemical group (carboxylic acids,
aldehydes, amines, sulfhydryls and hydroxyls) present on the
surface of the particle and present on the molecule to be
attached.
Functionality may be introduced into the particles in two ways. The
first is during the preparation of the nanoparticles, for example
during the emulsion preparation of nanoparticles by incorporation
of stabilizers with functional chemical groups. Suitable
stabilizers include hydrophobic or amphipathic molecules that
associate with the outer surface of the nanoparticles.
A second is post-particle preparation, by direct crosslinking
particles and ligands with homo- or heterobifunctional
crosslinkers. This second procedure may use a suitable chemistry
and a class of crosslinkers (CDI, EDAC, glutaraldehydes, etc. as
discussed in more detail below) or any other crosslinker that
couples ligands to the particle surface via chemical modification
of the particle surface after preparation. This second class also
includes a process whereby amphiphilic molecules such as fatty
acids, lipids or functional stabilizers may be passively adsorbed
and adhered to the particle surface, thereby introducing functional
end groups for tethering to ligands.
One useful protocol involves the "activation" of hydroxyl groups on
polymer chains with the agent, carbonyldiimidazole (CDI) in aprotic
solvents such as DMSO, acetone, or THF. CDI forms an imidazolyl
carbamate complex with the hydroxyl group which may be displaced by
binding the free amino group of a molecule such as a protein. The
reaction is an N-nucleophilic substitution and results in a stable
N-alkylcarbamate linkage of the molecule to the polymer. The
"coupling" of the molecule to the "activated" polymer matrix is
maximal in the pH range of 9-10 and normally requires at least 24
hrs. The resulting molecule-polymer complex is stable and resists
hydrolysis for extended periods of time.
Another coupling method involves the use of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or
"water-soluble CDI" in conjunction with N-hydroxylsulfosuccinimide
(sulfo NHS) to couple the exposed carboxylic groups of polymers to
the free amino groups of molecules in a totally aqueous environment
at the physiological pH of 7.0. Briefly, EDAC and sulfo-NHS form an
activated ester with the carboxylic acid groups of the polymer
which react with the amine end of a molecule to form a peptide
bond. The resulting peptide bond is resistant to hydrolysis. The
use of sulfo-NHS in the reaction increases the efficiency of the
EDAC coupling by a factor of ten-fold and provides for
exceptionally gentle conditions that ensure the viability of the
molecule-polymer complex.
By using either of these protocols it is possible to "activate"
almost all polymers containing either hydroxyl or carboxyl groups
in a suitable solvent system that will not dissolve the polymer
matrix.
A useful coupling procedure for attaching molecules with free
hydroxyl and carboxyl groups to polymers involves the use of the
cross-linking agent, divinylsulfone. This method would be useful
for attaching sugars or other hydroxylic compounds with bioadhesive
properties to hydroxylic matrices. Briefly, the activation involves
the reaction of divinylsulfone to the hydroxyl groups of the
polymer, forming the vinylsulfonyl ethyl ether of the polymer. The
vinyl groups will couple to alcohols, phenols and even amines.
Activation and coupling take place at pH 11. The linkage is stable
in the pH range from 1-8 and is suitable for transit through the
intestine.
Any suitable coupling method known to those skilled in the art for
the coupling of molecules and polymers with double bonds, including
the use of UV crosslinking, may be used for attachment of molecules
to the polymer.
In one embodiment, coupling agents can be conjugated to affinity
tags. Affinity tags are any molecular species which form highly
specific, noncovalent, physiochemical interactions with defined
binding partners. Affinity tags which form highly specific,
noncovalent, physiochemical interactions with one another are
defined herein as "complementary". Suitable affinity tag pairs are
well known in the art and include epitope/antibody, biotin/avidin,
biotin/streptavidin, biotin/neutravidin,
glutathione-S-transferase/glutathione, maltose binding
protein/amylase and maltose binding protein/maltose. Examples of
suitable epitopes which may be used for epitope/antibody binding
pairs include, but are not limited to, HA, FLAG, c-Myc,
glutatione-S-transferase, His.sub.6, GFP, DIG, biotin and avidin.
Antibodies (both monoclonal and polyclonal and antigen-binding
fragments thereof) which bind to these epitopes are well known in
the art.
Affinity tags that are conjugated to coupling agents allow for
highly flexible, modular assembly and disassembly of functional
elements which are conjugated to affinity tags which form highly
specific, noncovalent, physiochemical interactions with
complementary affinity tags which are conjugated to coupling
agents. Adaptor elements may be conjugated with a single species of
affinity tag or with any combination of affinity tag species in any
ratio. The ability to vary the number of species of affinity tags
and their ratios conjugated to adaptor elements allows for
exquisite control over the number of functional elements which may
be attached to the nanoparticles and their ratios.
In another embodiment, coupling agents are coupled directly to
functional elements in the absence of affinity tags, such as
through direct covalent interactions. Coupling agents can be
covalently coupled to at least one species of functional element.
Coupling agents can be covalently coupled to a single species of
functional element or with any combination of species of functional
elements in any ratio.
In a preferred embodiment, coupling agents are conjugated to at
least one affinity tag that provides for assembly and disassembly
of modular functional elements which are conjugated to
complementary affinity tags. In a more preferred embodiment,
coupling agents are fatty acids that are conjugated with at least
one affinity tag. In a particularly preferred embodiment, the
coupling agents are fatty acids conjugated with avidin or
streptavidin. Avidin/streptavidin-conjugated fatty acids allow for
the attachment of a wide variety of biotin-conjugated functional
elements.
The coupling agents are preferably provided on, or in the surface
of, nanoparticles at a high density. This high density of coupling
agents allows for coupling of the polymeric nanoparticles to a
variety of species of functional elements while still allowing for
the functional elements to be present in high enough numbers to be
efficacious.
1. Fatty Acids
The coupling agents may include fatty acids. Fatty acids may be of
any acyl chain length and may be saturated or unsaturated. In a
particularly preferred embodiment, the fatty acid is palmitic acid.
Other suitable fatty acids include, but are not limited to,
saturated fatty acids such as butyric, caproic, caprylic, capric,
lauric, myristic, stearic, arachidic and behenic acid. Still other
suitable fatty acids include, but are not limited to, unsaturated
fatty acids such as oleic, linoleic, alpha-linolenic, arachidonic,
eicosapentaenoic, docosahexaenoic and erucic acid.
2. Hydrophobic or Amphipathic Peptides
The coupling agents may include hydrophobic or amphipathic
peptides. Preferred peptides should be sufficiently hydrophobic to
preferentially associate with the polymeric nanoparticle over the
aqueous environment. Amphipathic polypeptides useful as adaptor
elements may be mostly hydrophobic on one end and mostly
hydrophilic on the other end. Such amphipathic peptides may
associate with polymeric nanoparticles through the hydrophobic end
of the peptide and be conjugated on the hydrophilic end to a
functional group.
3. Hydrophobic Polymers
Coupling agents may include hydrophobic polymers. Examples of
hydrophobic polymers include, but are not limited to,
polyanhydrides, poly(ortho)esters, and polyesters such as
polycaprolactone.
VII. Functional Molecules
Functional molecules can be associated with, linked, conjugated, or
otherwise attached directly or indirectly gene editing technology,
potentiating agents, or nanoparticles utilized for delivery
thereof.
A. Targeting Molecules
One class of functional elements is targeting molecules. Targeting
molecules can be associated with, linked, conjugated, or otherwise
attached directly or indirectly to the gene editing molecule, or to
a nanoparticle or other delivery vehicle thereof.
Targeting molecules can be proteins, peptides, nucleic acid
molecules, saccharides or polysaccharides that bind to a receptor
or other molecule on the surface of a targeted cell. The degree of
specificity and the avidity of binding to the graft can be
modulated through the selection of the targeting molecule. For
example, antibodies are very specific. These can be polyclonal,
monoclonal, fragments, recombinant, or single chain, many of which
are commercially available or readily obtained using standard
techniques.
Examples of moieties include, for example, targeting moieties which
provide for the delivery of molecules to specific cells, e.g.,
antibodies to hematopoietic stem cells, CD34.sup.+ cells, T cells
or any other preferred cell type, as well as receptor and ligands
expressed on the preferred cell type. Preferably, the moieties
target hematopoeitic stem cells.
Examples of molecules targeting extracellular matrix ("ECM")
include glycosaminoglycan ("GAG") and collagen. In one embodiment,
the external surface of polymer particles may be modified to
enhance the ability of the particles to interact with selected
cells or tissue. The method described above wherein an adaptor
element conjugated to a targeting molecule is inserted into the
particle is preferred. However, in another embodiment, the outer
surface of a polymer micro- or nanoparticle having a carboxy
terminus may be linked to targeting molecules that have a free
amine terminus.
Other useful ligands attached to polymeric micro- and nanoparticles
include pathogen-associated molecular patterns (PAMPs). PAMPs
target Toll-like Receptors (TLRs) on the surface of the cells or
tissue, or signal the cells or tissue internally, thereby
potentially increasing uptake. PAMPs conjugated to the particle
surface or co-encapsulated may include: unmethylated CpG DNA
(bacterial), double-stranded RNA (viral), lipopolysacharride
(bacterial), peptidoglycan (bacterial), lipoarabinomannin
(bacterial), zymosan (yeast), mycoplasmal lipoproteins such as
MALP-2 (bacterial), flagellin (bacterial) poly(inosinic-cytidylic)
acid (bacterial), lipoteichoic acid (bacterial) or
imidazoquinolines (synthetic).
In another embodiment, the outer surface of the particle may be
treated using a mannose amine, thereby mannosylating the outer
surface of the particle. This treatment may cause the particle to
bind to the target cell or tissue at a mannose receptor on the
antigen presenting cell surface. Alternatively, surface conjugation
with an immunoglobulin molecule containing an Fc portion (targeting
Fc receptor), heat shock protein moiety (HSP receptor),
phosphatidylserine (scavenger receptors), and lipopolysaccharide
(LPS) are additional receptor targets on cells or tissue.
Lectins that can be covalently attached to micro- and nanoparticles
to render them target specific to the mucin and mucosal cell layer
include lectins isolated from Abrus precatroius, Agaricus bisporus,
Anguilla anguilla, Arachis hypogaea, Pandeiraea simplicifolia,
Bauhinia purpurea, Caragan arobrescens, Cicer arietinum, Codium
fragile, Datura stramonium, Dolichos biflorus, Erythrina
corallodendron, Erythrina cristagalli, Euonymus europaeus, Glycine
max, Helix aspersa, Helix pomatia, Lathyrus odoratus, Lens
culinaris, Limulus polyphemus, Lysopersicon esculentum, Maclura
pomifera, Momordica charantia, Mycoplasma gallisepticum, Naja
mocambique, as well as the lectins Concanavalin A,
Succinyl-Concanavalin A, Triticum vulgaris, Ulex europaeus I, II
and III, Sambucus nigra, Maackia amurensis, Limax fluvus, Homarus
americanus, Cancer antennarius, and Lotus tetragonolobus.
The choice of targeting molecule will depend on the method of
administration of the nanoparticle composition and the cells or
tissues to be targeted. The targeting molecule may generally
increase the binding affinity of the particles for cell or tissues
or may target the nanoparticle to a particular tissue in an organ
or a particular cell type in a tissue. Avidin increases the ability
of polymeric nanoparticles to bind to tissues. While the exact
mechanism of the enhanced binding of avidin-coated particles to
tissues has not been elucidated, it is hypothesized it is caused by
electrostatic attraction of positively charged avidin to the
negatively charged extracellular matrix of tissue. Non-specific
binding of avidin, due to electrostatic interactions, has been
previously documented and zeta potential measurements of
avidin-coated PLGA particles revealed a positively charged surface
as compared to uncoated PLGA particles.
The attachment of any positively charged ligand, such as
polyethyleneimine or polylysine, to any polymeric particle may
improve bioadhesion due to the electrostatic attraction of the
cationic groups coating the beads to the net negative charge of the
mucus. The mucopolysaccharides and mucoproteins of the mucin layer,
especially the sialic acid residues, are responsible for the
negative charge coating. Any ligand with a high binding affinity
for mucin could also be covalently linked to most particles with
the appropriate chemistry and be expected to influence the binding
of particles to the gut. For example, polyclonal antibodies raised
against components of mucin or else intact mucin, when covalently
coupled to particles, would provide for increased bioadhesion.
Similarly, antibodies directed against specific cell surface
receptors exposed on the lumenal surface of the intestinal tract
would increase the residence time of beads, when coupled to
particles using the appropriate chemistry. The ligand affinity need
not be based only on electrostatic charge, but other useful
physical parameters such as solubility in mucin or else specific
affinity to carbohydrate groups.
The covalent attachment of any of the natural components of mucin
in either pure or partially purified form to the particles would
decrease the surface tension of the bead-gut interface and increase
the solubility of the bead in the mucin layer. The list of useful
ligands includes, but is not limited to the following: sialic acid,
neuraminic acid, n-acetyl-neuraminic acid, n-glycolylneuraminic
acid, 4-acetyl-n-acetylneuraminic acid, diacetyl-n-acetylneuraminic
acid, glucuronic acid, iduronic acid, galactose, glucose, mannose,
fucose, any of the partially purified fractions prepared by
chemical treatment of naturally occurring mucin, mucoproteins,
mucopolysaccharides and mucopolysaccharide-protein complexes, and
antibodies immunoreactive against proteins or sugar structure on
the mucosal surface.
The attachment of polyamino acids containing extra pendant
carboxylic acid side groups, e.g., polyaspartic acid and
polyglutamic acid, should also provide a useful means of increasing
bioadhesiveness. Using polyamino acids in the 15,000 to 50,000 kDa
molecular weight range yields chains of 120 to 425 amino acid
residues attached to the surface of the particles. The polyamino
chains increase bioadhesion by means of chain entanglement in mucin
strands as well as by increased carboxylic charge.
The efficacy of the nanoparticles is determined in part by their
route of administration into the body. For orally and topically
administered nanoparticles, epithelial cells constitute the
principal barrier that separates an organism's interior from the
outside world. Epithelial cells such as those that line the
gastrointestinal tract form continuous monolayers that
simultaneously confront the extracellular fluid compartment and the
extracorporeal space.
Adherence to cells is an essential first step in crossing the
epithelial barrier by any of these mechanisms. Therefore, in one
embodiment, the nanoparticles disclosed herein further include
epithelial cell targeting molecules. Epithelial cell targeting
molecules include monoclonal or polyclonal antibodies or bioactive
fragments thereof that recognize and bind to epitopes displayed on
the surface of epithelial cells. Epithelial cell targeting
molecules also include ligands which bind to a cell surface
receptor on epithelial cells. Ligands include, but are not limited
to, molecules such as polypeptides, nucleotides and
polysaccharides.
A variety of receptors on epithelial cells may be targeted by
epithelial cell targeting molecules. Examples of suitable receptors
to be targeted include, but are not limited to, IgE Fc receptors,
EpCAM, selected carbohydrate specificites, dipeptidyl peptidase,
and E-cadherin.
B. Protein Transduction Domains and Fusogenic Peptides
Other functional elements that can be associated with, linked,
conjugated, or otherwise attached directly or indirectly to the
gene editing molecule, potentiating agent, or to a nanoparticle or
other delivery vehicle thereof, include protein transduction
domains and fusogenic peptides.
For example, the efficiency of nanoparticle delivery systems can
also be improved by the attachment of functional ligands to the NP
surface. Potential ligands include, but are not limited to, small
molecules, cell-penetrating peptides (CPPs), targeting peptides,
antibodies or aptamers (Yu, et al., PLoS One., 6:e24077 (2011), Cu,
et al., J Control Release, 156:258-264 (2011), Nie, et al., J
Control Release, 138:64-70 (2009), Cruz, et al., J Control Release,
144:118-126 (2010)). Attachment of these moieties serves a variety
of different functions; such as inducing intracellular uptake,
endosome disruption, and delivery of the plasmid payload to the
nucleus. There have been numerous methods employed to tether
ligands to the particle surface. One approach is direct covalent
attachment to the functional groups on PLGA NPs (Bertram, Acta
Biomater. 5:2860-2871 (2009)). Another approach utilizes
amphiphilic conjugates like avidin palmitate to secure biotinylated
ligands to the NP surface (Fahmy, et al., Biomaterials,
26:5727-5736 (2005), Cu, et al., Nanomedicine, 6:334-343 (2010)).
This approach produces particles with enhanced uptake into cells,
but reduced pDNA release and gene transfection, which is likely due
to the surface modification occluding pDNA release. In a similar
approach, lipid-conjugated polyethylene glycol (PEG) is used as a
multivalent linker of penetratin, a CPP, or folate (Cheng, et al.,
Biomaterials, 32:6194-6203 (2011)).
These methods, as well as other methods discussed herein, and
others methods known in the art, can be combined to tune particle
function and efficacy. In some preferred embodiments, PEG is used
as a linker for linking functional molecules to nanoparticles. For
example, DSPE-PEG(2000)-maleimide is commercially available and can
be used utilized for covalently attaching functional molecules such
as CPP.
"Protein Transduction Domain" or PTD refers to a polypeptide,
polynucleotide, or organic or inorganic compounds that facilitates
traversing a lipid bilayer, micelle, cell membrane, organelle
membrane, or vesicle membrane. A PTD attached to another molecule
facilitates the molecule traversing membranes, for example going
from extracellular space to intracellular space, or cytosol to
within an organelle. PTA can be short basic peptide sequences such
as those present in many cellular and viral proteins. Exemplary
protein transduction domains that are well-known in the art
include, but are not limited to, the Antennapedia PTD and the TAT
(transactivator of transcription) PTD, poly-arginine, poly-lysine
or mixtures of arginine and lysine, HIV TAT (YGRKKRRQRRR (SEQ ID
NO:7) or RKKRRQRRR (SEQ ID NO:8), 11 arginine residues, VP22
peptide, and an ANTp peptide (RQIKIWFQNRRMKWKK) (SEQ ID NO:9) or
positively charged polypeptides or polynucleotides having 8-15
residues, preferably 9-11 residues. Short, non-peptide polymers
that are rich in amines or guanidinium groups are also capable of
carrying molecules crossing biological membranes. Penetratin and
other derivatives of peptides derived from antennapedia (Cheng, et
al., Biomaterials, 32(26):6194-203 (2011) can also be used. Results
show that penetratin in which additional Args are added, further
enhances uptake and endosomal escape, and IKK NBD, which has an
antennapedia domain for permeation as well as a domain that blocks
activation of NFkB and has been used safely in the lung for other
purposes (von Bismarck, et al., Pulmonary Pharmacology &
Therapeutics, 25(3):228-35 (2012), Kamei, et al., Journal Of
Pharmaceutical Sciences, 102(11):3998-4008 (2013)).
A "fusogenic peptide" is any peptide with membrane destabilizing
abilities. In general, fusogenic peptides have the propensity to
form an amphiphilic alpha-helical structure when in the presence of
a hydrophobic surface such as a membrane. The presence of a
fusogenic peptide induces formation of pores in the cell membrane
by disruption of the ordered packing of the membrane phospholipids.
Some fusogenic peptides act to promote lipid disorder and in this
way enhance the chance of merging or fusing of proximally
positioned membranes of two membrane enveloped particles of various
nature (e.g. cells, enveloped viruses, liposomes). Other fusogenic
peptides may simultaneously attach to two membranes, causing
merging of the membranes and promoting their fusion into one.
Examples of fusogenic peptides include a fusion peptide from a
viral envelope protein ectodomain, a membrane-destabilizing peptide
of a viral envelope protein membrane-proximal domain from the
cytoplasmic tails.
Other fusogenic peptides often also contain an amphiphilic-region.
Examples of amphiphilic-region containing peptides include:
melittin, magainins, the cytoplasmic tail of HIV1 gp41, microbial
and reptilian cytotoxic peptides such as bomolitin 1, pardaxin,
mastoparan, crabrolin, cecropin, entamoeba, and staphylococcal
.alpha.-toxin; viral fusion peptides from (1) regions at the N
terminus of the transmembrane (TM) domains of viral envelope
proteins, e.g. HIV-1, SIV, influenza, polio, rhinovirus, and
coxsackie virus; (2) regions internal to the TM ectodomain, e.g.
semliki forest virus, sindbis virus, rota virus, rubella virus and
the fusion peptide from sperm protein PH-30: (3) regions
membrane-proximal to the cytoplasmic side of viral envelope
proteins e.g. in viruses of avian leukosis (ALV), Feline
immunodeficiency (FIV), Rous Sarcoma (RSV), Moloney murine leukemia
virus (MoMuLV), and spleen necrosis (SNV).
In particular embodiments, a functional molecule such as a CPP is
covalently linked to DSPE-PEG-maleimide functionalized
nanoparticles such as PBAE/PLGA blended particles using known
methods such as those described in Fields, et al., J Control
Release, 164(1):41-48 (2012). For example, DSPE-PEG-function
molecule can be added to the 5.0% PVA solution during formation of
the second emulsion. In some embodiments, the loading ratio is
about 5 nmol/mg ligand-to-polymer ratio.
In some embodiments, the functional molecule is a CPP such as those
above, or mTAT (HIV-1 (with histidine modification)
HHHHRKKRRQRRRRHHHHH (SEQ ID NO:10) (Yamano, et al., J Control
Release, 152:278-285 (2011)); or bPrPp (Bovine prion)
MVKSKIGSWILVLFVAMWS DVGLCKKRPKP (SEQ ID NO:11) (Magzoub, et al.,
Biochem Biophys Res Commun., 348:379-385 (2006)); or MPG (Synthetic
chimera: SV40 Lg T. Ant.+ HIV gb41 coat) GALFLGFLGAAGSTMGAWS
QPKKKRKV (SEQ ID NO:12) (Endoh, et al., Adv Drug Deliv Rev.,
61:704-709 (2009)).
VIII. Methods of Manufacture
A. Methods of Making Nanoparticles
The nanoparticle compositions described herein can be prepared by a
variety of methods.
1. Polycations
In some embodiments, the nucleic acid is first complexed to a
polycation. Complexation can be achieved by mixing the nucleic
acids and polycations at an appropriate molar ratio. When a
polyamine is used as the polycation species, it is useful to
determine the molar ratio of the polyamine nitrogen to the
polynucleotide phosphate (N/P ratio). In a preferred embodiment,
nucleic acids and polyamines are mixed together to form a complex
at an N/P ratio of between approximately 8:1 to 15:1. The volume of
polyamine solution required to achieve particular molar ratios can
be determined according to the following formula:
.times..times..times..times..times..PHI..times..PHI..times..times..times.-
.times..times..times. ##EQU00001## where M.sub.w,nucacid=molecular
weight of nucleic acid, M.sub.w,P=molecular weight of phosphate
groups of the nucleic acid, .PHI..sub.N:P=N:P ratio (molar ratio of
nitrogens from polyamine to the ratio of phosphates from the
nucleic acid), C.sub.NH2, stock=concentration of polyamine stock
solution, and M.sub.w,NH2=molecular weight per nitrogen of
polyamine.
Polycation complexation with nucleic acids can be achieved by
mixing solutions containing polycations with solutions containing
nucleic acids. The mixing can occur at any appropriate temperature.
In one embodiment, the mixing occurs at room temperature. The
mixing can occur with mild agitation, such as can be achieved
through the use of a rotary shaker.
2. Exemplary Preferred Methods of Manufacture
In preferred embodiments, the nanoparticles are formed by a
double-emulsion solvent evaporation technique, such as is disclosed
in U.S. Published Application No. 2011/0008451 or U.S. Published
Application No. 2011/0268810, each of which is a specifically
incorporated by reference in its entirety, or Fahmy, et al.,
Biomaterials, 26:5727-5736, (2005), or McNeer, et al., Mol. Ther.
19, 172-180 (2011)). In this technique, the nucleic acids or
nucleic acid/polycation complexes are reconstituted in an aqueous
solution. Nucleic acid and polycation amounts are discussed in more
detail below and can be chosen, for example, based on amounts and
ratios disclosed in U.S. Published Application No. 2011/0008451 or
U.S. Published Application No. 2011/0268810, or used by McNeer, et
al., (McNeer, et al., Mol. Ther. 19, 172-180 (2011)), or by Woodrow
et al. for small interfering RNA encapsulation (Woodrow, et al.,
Nat Mater, 8:526-533 (2009)). This aqueous solution is then added
dropwise to a polymer solution of a desired polymer dissolved in an
organic solvent to form the first emulsion.
This mixture is then added dropwise to solution containing a
surfactant, such as polyvinyl alcohol (PVA) and sonicated to form
the double emulsion. The final emulsion is then poured into a
solution containing the surfactant in an aqueous solution and
stirred for a period of time to allow the dichloromethane to
evaporate and the particles to harden. The concentration of the
surfactant used to form the emulsion, and the sonication time and
amplitude can been optimized according to principles known in the
art for formulating particles with a desired diameter. The
particles can be collected by centrifugation. If it is desirable to
store the nanoparticles for later use, they can be rapidly frozen,
and lyophilized.
In preferred embodiments the nanoparticles are PLGA nanoparticles.
In a particular exemplary protocol, nucleic acid (such as PNA, DNA,
or PNA-DNA) with or without a polycation (such as spermidine) are
dissolved in DNAse/RNAse free H.sub.2O. Encapsulant in H.sub.2O can
be added dropwise to a polymer solution of 50:50 ester-terminated
PLGA dissolved in dichloromethane (DCM), then sonicated to form the
first emulsion. This emulsion can then be added dropwise to 5%
polyvinyl alcohol, then sonicated to form the second emulsion. This
mixture can be poured into 0.3% polyvinyl alcohol, and stirred at
room temperature to form nanoparticles. Nanoparticles can then be
collected and washed with, for example H.sub.2O, collected by
centrifugation, and then resuspended in H.sub.2O, frozen at
-80.degree. C., and lyophilized. Particles can be stored at
-20.degree. C. following lyophilization.
Additional techniques for encapsulating the nucleic acid and
polycation complex into polymeric nanoparticles are described
below.
3. Solvent Evaporation
In this method the polymer is dissolved in a volatile organic
solvent, such as methylene chloride. The drug (either soluble or
dispersed as fine particles) is added to the solution, and the
mixture is suspended in an aqueous solution that contains a surface
active agent such as poly(vinyl alcohol). The resulting emulsion is
stirred until most of the organic solvent evaporated, leaving solid
particles. The resulting particles are washed with water and dried
overnight in a lyophilizer Particles with different sizes (0.5-1000
microns) and morphologies can be obtained by this method. This
method is useful for relatively stable polymers like polyesters and
polystyrene.
However, labile polymers, such as polyanhydrides, may degrade
during the fabrication process due to the presence of water. For
these polymers, the following two methods, which are performed in
completely anhydrous organic solvents, are more useful.
4. Interfacial Polycondensation
Interfacial polycondensation is used to microencapsulate a core
material in the following manner. One monomer and the core material
are dissolved in a solvent. A second monomer is dissolved in a
second solvent (typically aqueous) which is immiscible with the
first. An emulsion is formed by suspending the first solution
through stirring in the second solution. Once the emulsion is
stabilized, an initiator is added to the aqueous phase causing
interfacial polymerization at the interface of each droplet of
emulsion.
5. Solvent Evaporation Microencapsulation
In solvent evaporation microencapsulation, the polymer is typically
dissolved in a water immiscible organic solvent and the material to
be encapsulated is added to the polymer solution as a suspension or
solution in an organic solvent. An emulsion is formed by adding
this suspension or solution to a beaker of vigorously stirring
water (often containing a surface active agent, for example,
polyethylene glycol or polyvinyl alcohol, to stabilize the
emulsion). The organic solvent is evaporated while continuing to
stir. Evaporation results in precipitation of the polymer, forming
solid microcapsules containing core material.
The solvent evaporation process can be used to entrap a liquid core
material in a polymer such as PLA, PLA/PGA copolymer, or PLA/PCL
copolymer microcapsules. The polymer or copolymer is dissolved in a
miscible mixture of solvent and nonsolvent, at a nonsolvent
concentration which is immediately below the concentration which
would produce phase separation (i.e., cloud point). The liquid core
material is added to the solution while agitating to form an
emulsion and disperse the material as droplets. Solvent and
nonsolvent are vaporized, with the solvent being vaporized at a
faster rate, causing the polymer or copolymer to phase separate and
migrate towards the surface of the core material droplets. This
phase-separated solution is then transferred into an agitated
volume of nonsolvent, causing any remaining dissolved polymer or
copolymer to precipitate and extracting any residual solvent from
the formed membrane. The result is a microcapsule composed of
polymer or copolymer shell with a core of liquid material.
Solvent evaporation microencapsulation can result in the
stabilization of insoluble active agent particles in a polymeric
solution for a period of time ranging from 0.5 hours to several
months. Stabilizing an insoluble pigment and polymer within the
dispersed phase (typically a volatile organic solvent) can be
useful for most methods of microencapsulation that are dependent on
a dispersed phase, including film casting, solvent evaporation,
solvent removal, spray drying, phase inversion, and many
others.
The stabilization of insoluble active agent particles within the
polymeric solution could be critical during scale-up. By
stabilizing suspended active agent particles within the dispersed
phase, the particles can remain homogeneously dispersed throughout
the polymeric solution as well as the resulting polymer matrix that
forms during the process of microencapsulation.
Solvent evaporation microencapsulation (SEM) have several
advantages. SEM allows for the determination of the best
polymer-solvent-insoluble particle mixture that will aid in the
formation of a homogeneous suspension that can be used to
encapsulate the particles. SEM stabilizes the insoluble particles
or pigments within the polymeric solution, which will help during
scale-up because one will be able to let suspensions of insoluble
particles or pigments sit for long periods of time, making the
process less time-dependent and less labor intensive. SEM allows
for the creation of nanoparticles that have a more optimized
release of the encapsulated material.
6. Hot Melt Microencapsulation
In this method, the polymer is first melted and then mixed with the
solid particles. The mixture is suspended in a non-miscible solvent
(like silicon oil), and, with continuous stirring, heated to
5.degree. C. above the melting point of the polymer. Once the
emulsion is stabilized, it is cooled until the polymer particles
solidify. The resulting particles are washed by decantation with
petroleum ether to give a free-flowing powder. Particles with sizes
between 0.5 to 1000 microns are obtained with this method. The
external surfaces of spheres prepared with this technique are
usually smooth and dense. This procedure is used to prepare
particles made of polyesters and polyanhydrides. However, this
method is limited to polymers with molecular weights between
1,000-50,000.
7. Solvent Removal Microencapsulation
In solvent removal microencapsulation, the polymer is typically
dissolved in an oil miscible organic solvent and the material to be
encapsulated is added to the polymer solution as a suspension or
solution in organic solvent. Surface active agents can be added to
improve the dispersion of the material to be encapsulated. An
emulsion is formed by adding this suspension or solution to
vigorously stirring oil, in which the oil is a nonsolvent for the
polymer and the polymer/solvent solution is immiscible in the oil.
The organic solvent is removed by diffusion into the oil phase
while continuing to stir. Solvent removal results in precipitation
of the polymer, forming solid microcapsules containing core
material.
8. Phase Separation Microencapsulation
In phase separation microencapsulation, the material to be
encapsulated is dispersed in a polymer solution with stirring.
While continually stirring to uniformly suspend the material, a
nonsolvent for the polymer is slowly added to the solution to
decrease the polymer's solubility. Depending on the solubility of
the polymer in the solvent and nonsolvent, the polymer either
precipitates or phase separates into a polymer rich and a polymer
poor phase. Under proper conditions, the polymer in the polymer
rich phase will migrate to the interface with the continuous phase,
encapsulating the core material in a droplet with an outer polymer
shell.
9. Spontaneous Emulsification
Spontaneous emulsification involves solidifying emulsified liquid
polymer droplets by changing temperature, evaporating solvent, or
adding chemical cross-linking agents. The physical and chemical
properties of the encapsulant, and the material to be encapsulated,
dictates the suitable methods of encapsulation. Factors such as
hydrophobicity, molecular weight, chemical stability, and thermal
stability affect encapsulation.
10. Coacervation
Encapsulation procedures for various substances using coacervation
techniques have been described in the prior art, for example, in
GB-B-929 406; GB-B-929 401; U.S. Pat. Nos. 3,266,987; 4,794,000 and
4,460,563. Coacervation is a process involving separation of
colloidal solutions into two or more immiscible liquid layers (Ref.
Dowben, R. General Physiology, Harper & Row, New York, 1969,
pp. 142-143.). Through the process of coacervation compositions
comprised of two or more phases and known as coacervates may be
produced. The ingredients that comprise the two phase coacervate
system are present in both phases; however, the colloid rich phase
has a greater concentration of the components than the colloid poor
phase.
11. Solvent Removal
This technique is primarily designed for polyanhydrides. In this
method, the drug is dispersed or dissolved in a solution of the
selected polymer in a volatile organic solvent like methylene
chloride. This mixture is suspended by stirring in an organic oil
(such as silicon oil) to form an emulsion. Unlike solvent
evaporation, this method can be used to make particles from
polymers with high melting points and different molecular weights.
Particles that range between 1-300 microns can be obtained by this
procedure. The external morphology of spheres produced with this
technique is highly dependent on the type of polymer used.
12. Spray-Drying
In this method, the polymer is dissolved in organic solvent. A
known amount of the active drug is suspended (insoluble drugs) or
co-dissolved (soluble drugs) in the polymer solution. The solution
or the dispersion is then spray-dried. Typical process parameters
for a mini-spray drier (Buchi) are as follows: polymer
concentration=0.04 g/mL, inlet temperature=-24.degree. C., outlet
temperature=13-15.degree. C., aspirator setting=15, pump setting=10
mL/minute, spray flow=600 Nl/hr, and nozzle diameter=0.5 mm.
Particles ranging between 1-10 microns are obtained with a
morphology which depends on the type of polymer used.
13. Nanoprecipitation
In nanoprecipitation, the polymer and nucleic acids are
co-dissolved in a selected, water-miscible solvent, for example
DMSO, acetone, ethanol, acetone, etc. In a preferred embodiment,
nucleic acids and polymer are dissolved in DMSO. The solvent
containing the polymer and nucleic acids is then drop-wise added to
an excess volume of stirring aqueous phase containing a stabilizer
(e.g., poloxamer, Pluronic.RTM., and other stabilizers known in the
art). Particles are formed and precipitated during solvent
evaporation. To reduce the loss of polymer, the viscosity of the
aqueous phase can be increased by using a higher concentration of
the stabilizer or other thickening agents such as glycerol and
others known in the art. Lastly, the entire dispersed system is
centrifuged, and the nucleic acid-loaded polymer nanoparticles are
collected and optionally filtered. Nanoprecipitation-based
techniques are discussed in, for example, U.S. Pat. No.
5,118,528.
Advantages to nanoprecipitation include: the method can
significantly increase the encapsulation efficiency of drugs that
are polar yet water-insoluble, compared to single or double
emulsion methods (Alshamsan, Saudi Pharmaceutical Journal,
22(3):219-222 (2014)). No emulsification or high shear force step
(e.g., sonication or high-speed homogenization) is involved in
nanoprecipitation, therefore preserving the conformation of nucleic
acids. Nanoprecipitation relies on the differences in the
interfacial tension between the solvent and the nonsolvent, rather
than shear stress, to produce nanoparticles. Hydrophobicity of the
drug will retain it in the instantly-precipitating nanoparticles;
the un-precipitated polymer due to equilibrium is "lost" and not in
the precipitated nanoparticle form.
B. Molecules to be Encapsulated or Attached to the Surface of the
Particles
There are two principle groups of molecules to be encapsulated or
attached to the polymer, either directly or via a coupling
molecule: targeting molecules, attachment molecules and
therapeutic, nutritional, diagnostic or prophylactic agents. These
can be coupled using standard techniques. The targeting molecule or
therapeutic molecule to be delivered can be coupled directly to the
polymer or to a material such as a fatty acid which is incorporated
into the polymer.
Functionality refers to conjugation of a ligand to the surface of
the particle via a functional chemical group (carboxylic acids,
aldehydes, amines, sulfhydryls and hydroxyls) present on the
surface of the particle and present on the ligand to be attached.
Functionality may be introduced into the particles in two ways. The
first is during the preparation of the particles, for example
during the emulsion preparation of particles by incorporation of
stabilizers with functional chemical groups. Example 1 demonstrates
this type of process whereby functional amphiphilic molecules are
inserted into the particles during emulsion preparation.
A second is post-particle preparation, by direct crosslinking
particles and ligands with homo- or heterobifunctional
crosslinkers. This second procedure may use a suitable chemistry
and a class of crosslinkers (CDI, EDAC, glutaraldehydes, etc. as
discussed in more detail below) or any other crosslinker that
couples ligands to the particle surface via chemical modification
of the particle surface after preparation. This second class also
includes a process whereby amphiphilic molecules such as fatty
acids, lipids or functional stabilizers may be passively adsorbed
and adhered to the particle surface, thereby introducing functional
end groups for tethering to ligands.
In the preferred embodiment, the surface is modified to insert
amphiphilic polymers or surfactants that match the polymer phase
HLB or hydrophile-lipophile balance, as demonstrated in the
following example. HLBs range from 1 to 15. Surfactants with a low
HLB are more lipid loving and thus tend to make a water in oil
emulsion while those with a high HLB are more hydrophilic and tend
to make an oil in water emulsion. Fatty acids and lipids have a low
HLB below 10. After conjugation with target group (such as
hydrophilic avidin), HLB increases above 10. This conjugate is used
in emulsion preparation. Any amphiphilic polymer with an HLB in the
range 1-10, more preferably between 1 and 6, most preferably
between 1 and up to 5, can be used. This includes all lipids, fatty
acids and detergents.
One useful protocol involves the "activation" of hydroxyl groups on
polymer chains with the agent, carbonyldiimidazole (CDI) in aprotic
solvents such as DMSO, acetone, or THF. CDI forms an imidazolyl
carbamate complex with the hydroxyl group which may be displaced by
binding the free amino group of a ligand such as a protein. The
reaction is an N-nucleophilic substitution and results in a stable
N-alkylcarbamate linkage of the ligand to the polymer. The
"coupling" of the ligand to the "activated" polymer matrix is
maximal in the pH range of 9-10 and normally requires at least 24
hrs. The resulting ligand-polymer complex is stable and resists
hydrolysis for extended periods of time.
Another coupling method involves the use of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) or
"water-soluble CDI" in conjunction with N-hydroxylsulfosuccinimide
(sulfo NHS) to couple the exposed carboxylic groups of polymers to
the free amino groups of ligands in a totally aqueous environment
at the physiological pH of 7.0. Briefly, EDAC and sulfo-NHS form an
activated ester with the carboxylic acid groups of the polymer
which react with the amine end of a ligand to form a peptide bond.
The resulting peptide bond is resistant to hydrolysis. The use of
sulfo-NHS in the reaction increases the efficiency of the EDAC
coupling by a factor of ten-fold and provides for exceptionally
gentle conditions that ensure the viability of the ligand-polymer
complex.
By using either of these protocols it is possible to "activate"
almost all polymers containing either hydroxyl or carboxyl groups
in a suitable solvent system that will not dissolve the polymer
matrix.
A useful coupling procedure for attaching ligands with free
hydroxyl and carboxyl groups to polymers involves the use of the
cross-linking agent, divinylsulfone. This method would be useful
for attaching sugars or other hydroxylic compounds with bioadhesive
properties to hydroxylic matrices. Briefly, the activation involves
the reaction of divinylsulfone to the hydroxyl groups of the
polymer, forming the vinylsulfonyl ethyl ether of the polymer. The
vinyl groups will couple to alcohols, phenols and even amines.
Activation and coupling take place at pH 11. The linkage is stable
in the pH range from 1-8 and is suitable for transit through the
intestine.
Any suitable coupling method known to those skilled in the art for
the coupling of ligands and polymers with double bonds, including
the use of UV crosslinking, may be used for attachment of molecules
to the polymer.
Coupling is preferably by covalent binding but it may also be
indirect, for example, through a linker bound to the polymer or
through an interaction between two molecules such as strepavidin
and biotin. It may also be by electrostatic attraction by
dip-coating.
The molecules to be delivered can also be encapsulated into the
polymer using double emulsion solvent evaporation techniques, such
as that described by Luo et al., Controlled DNA delivery system,
Phar. Res., 16: 1300-1308 (1999).
C. Particularly Preferred Nanoparticle Formulations
The nanoparticle formulation can be selected based on the
considerations including the targeted tissue or cells. For example,
in embodiments directed to treatment of treating or correcting
beta-thalassemia (e.g. when the target cells are, for example,
hematopoietic stem cells), a preferred nanoparticle formulation is
PLGA.
Other preferred nanoparticle formulations, particularly preferred
for treating cystic fibrosis, are described in McNeer, et al.,
Nature Commun., 6:6952. doi: 10.1038/ncomms7952 (2015), and Fields,
et al., Adv Healthc Mater., 4(3):361-6 (2015). doi:
10.1002/adhm.201400355 (2015) Epub 2014. Such nanoparticles are
composed of a blend of Poly(beta-amino) esters (PBAEs) and
poly(lactic-co-glycolic acid) (PLGA). Poly(beta-amino) esters
(PBAEs) are degradable, cationic polymers synthesized by conjugate
(Michael-like) addition of bifunctional amines to diacrylate esters
(Lynn, Langer R, editor. J Am Chem Soc. 2000. pp. 10761-10768).
PBAEs appear to have properties that make them efficient vectors
for gene delivery. These cationic polymers are able to condense
negatively charged pDNA, induce cellular uptake, and buffer the low
pH environment of endosomes leading to DNA escape (Lynn, Langer R,
editor. J Am Chem Soc. 2000. pp. 10761-10768, and Green, Acc Chem
Res., 41(6):749-759 (2008)). PBAEs have the ability to form hybrid
particles with other polymers, which allows for production of
solid, stable and storable particles. For example, blending
cationic PBAE with PLGA produced highly loaded pDNA particles. The
addition of PBAE to PLGA resulted in an increase in gene
transfection in vitro and induced antigen-specific tumor rejection
in a murine model (Little, et al. Proc Natl Acad Sci USA.,
101:9534-9539 (2004), Little, et al., J Control Release,
107:449-462 (2005)).
Therefore, in some embodiments, the nanoparticles utilized to
deliver the disclosed compositions are composed of a blend of PBAE
and a second polymer one of those discussed above. In some
embodiments, the nanoparticles are composed of a blend of PBAE and
PLGA.
PLGA and PBAE/PLGA blended nanoparticles loaded with gene editing
technology can be formulated using a double-emulsion solvent
evaporation technique such as that described in detail above, and
in McNeer, et al., Nature Commun., 6:6952. doi: 10.1038/ncomms7952
(2015), and Fields, et al., Adv Healthc Mater., 4(3):361-6 (2015).
doi: 10.1002/adhm.201400355 (2015) Epub 2014. Poly(beta amino
ester) (PBAE) can synthesized by a Michael addition reaction of
1,4-butanediol diacrylate and 4,4'-trimethylenedipiperidine as
described in Akinc, et al., Bioconjug Chem., 14:979-988 (2003). In
some embodiments, PBAE blended particles such as PLGA/PBAE blended
particles, contain between about 1 and 99, or between about 1 and
50, or between about 5 and 25, or between about 5 and 20, or
between about 10 and 20, or about 15 percent PBAE (wt %). In
particular embodiments, PBAE blended particles such as PLGA/PBAE
blended particles, contain about 50, 45, 40, 35, 30, 25, 20, 15,
10, or 5% PBAE (wt %). Solvent from these particles in PVA as
discussed above, and in some cases may continue overnight.
PLGA/PBAE/MPG nanoparticles was shown to produce significantly
greater nanoparticle association with airway epithelial cells than
PLGA nanoparticles (Fields, et al., Advanced Healthcare Materials,
4:361-366 (2015)).
IX. Methods of Use
A. Methods of Treatment
The disclosed compositions can be used to ex vivo or in vivo gene
editing. The methods typically include contacting a cell with an
effective amount of gene editing composition, preferably in
combination with a potentiating agent, to modify the cell's genome.
As discussed in more detail below, the contacting can occur ex vivo
or in vivo. In preferred embodiments, the method includes
contacting a population of target cells with an effective amount of
gene editing composition, preferably in combination with a
potentiating agent, to modify the genomes of a sufficient number of
cells to achieve a therapeutic result.
For example, the effective amount or therapeutically effective
amount can be a dosage sufficient to treat, inhibit, or alleviate
one or more symptoms of a disease or disorder, or to otherwise
provide a desired pharmacologic and/or physiologic effect, for
example, reducing, inhibiting, or reversing one or more of the
underlying pathophysiological mechanisms underlying a disease or
disorder.
In some embodiments, when the gene editing technology is triplex
forming molecules, the molecules can be administered in an
effective amount to induce formation of a triple helix at the
target site. An effective amount of gene editing technology such as
triplex-forming molecules may also be an amount effective to
increase the rate of recombination of a donor fragment relative to
administration of the donor fragment in the absence of the gene
editing technology. The formulation is made to suit the mode of
administration. Pharmaceutically acceptable carriers are determined
in part by the particular composition being administered, as well
as by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions containing the nucleic acids. The
precise dosage will vary according to a variety of factors such as
subject-dependent variables (e.g., age, immune system health,
clinical symptoms etc.). Exemplary symptoms, pharmacologic, and
physiologic effects are discussed in more detail below.
The disclosed compositions can be administered or otherwise
contacted with target cells once, twice, or three time daily; one,
two, three, four, five, six, seven times a week, one, two, three,
four, five, six, seven or eight times a month. For example, in some
embodiments, the composition is administered every two or three
days, or on average about 2 to about 4 times about week.
In some embodiments, the potentiating agent is administered to the
subject prior to administration of the gene editing technology to
the subject. The potentiating agent can be administered to the
subject, for example, 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours,
or 1, 2, 3, 4, 5, 6, or 7 days, or any combination thereof prior to
administration of the gene editing technology to the subject.
In some embodiments, the gene editing technology is administered to
the subject prior to administration of the potentiating agent to
the subject. The gene editing technology can be administered to the
subject, for example, 1, 2, 3, 4, 5, 6, 8, 10, 12, 18, or 24 hours,
or 1, 2, 3, 4, 5, 6, or 7 days, or any combination thereof prior to
administration of the potentiating agent to the subject.
In preferred embodiments, the compositions are administered in an
amount effective to induce gene modification in at least one target
allele to occur at frequency of at least 0.1, 0.2. 0.3, 0.4, 0.5,
0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25% of target cells. In
some embodiments, particularly ex vivo applications, gene
modification occurs in at least one target allele at a frequency of
about 0.1-25%, or 0.5-25%, or 1-25% 2-25%, or 3-25%, or 4-25% or
5-25% or 6-25%, or 7-25%, or 8-25%, or 9-25%, or 10-25%, 11-25%, or
12-25%, or 13%-25% or 14%-25% or 15-25%, or 2-20%, or 3-20%, or
4-20% or 5-20% or 6-20%, or 7-20%, or 8-20%, or 9-20%, or 10-20%,
11-20%, or 12-20%, or 13%-20% or 14%-20% or 15-20%, 2-15%, or
3-15%, or 4-15% or 5-15% or 6-15%, or 7-15%, or 8-15%, or 9-15%, or
10-15%, 11-15%, or 12-15%, or 13%-15% or 14%-15%.
In some embodiments, particularly in vivo applications, gene
modification occurs in at least one target allele at a frequency of
about 0.1% to about 10%, or about 0.2% to about 10%, or about 0.3%
to about 10%, or about 0.4% to about 10%, or about 0.5% to about
10%, or about 0.6% to about 10%, or about 0.7% to about 10%, or
about 0.8% to about 10%, or about 0.9% to about 10%, or about 1.0%
to about 10%, or about 1.1% to about 10%, or about 1.1% to about
10%, 1.2% to about 10%, or about 1.3% to about 10%, or about 1.4%
to about 10%, or about 1.5% to about 10%, or about 1.6% to about
10%, or about 1.7% to about 10%, or about 1.8% to about 10%, or
about 1.9% to about 10%, or about 2.0% to about 10%, or about 2.5%
to about 10%, or about 3.0% to about 10%, or about 3.5% to about
10%, or about 4.0% to about 10%, or about 4.5% to about 10%, or
about 5.0% to about 10%.
In some embodiments, gene modification occurs with low off-target
effects. In some embodiments, off-target modification is
undetectable using routine analysis such as those described in the
Examples below. In some embodiments, off-target incidents occur at
a frequency of 0-1%, or 0-0.1%, or 0-0.01%, or 0-0.001%, or
0-0.0001%, or 0-0000.1%, or 0-0.000001%. In some embodiments,
off-target modification occurs at a frequency that is about
10.sup.2, 10.sup.3, 10.sup.4, or 10.sup.5-fold lower than at the
target site.
Gene Editing Technology
In general, by way of example only, dosage forms useful in the
disclosed methods can include doses in the range of about 10.sup.2
to about 10.sup.50, or about 10.sup.5 to about 10.sup.40, or about
10.sup.10 to about 10.sup.30, or about 10.sup.12 to about 10.sup.20
copies of the gene editing technology per dose. In particular
embodiments, about 10.sup.13, 10.sup.14, 10.sup.15, 10.sup.16, or
10.sup.17 copies of gene editing technology are administered to a
subject in need thereof.
In other embodiments, dosages are expressed in moles. For example,
in some embodiments, the dose of gene editing technology is about
0.1 nmol to about 100 nmol, or about 0.25 nmol to about 50 nmol, or
about 0.5 nmol to about 25 nmol, or about 0.75 nmol to about 7.5
nmol.
In other embodiments, dosages are expressed in molecules per target
cells. For example, in some embodiments, the dose of gene editing
technology is about 10.sup.2 to about 10.sup.50, or about 10.sup.5
to about 10.sup.15, or about 10.sup.7 to about 10.sup.12, or about
10.sup.8 to about 10.sup.11 copies of the gene editing technology
per target cell.
In other embodiments, dosages are expressed in mg/kg, particularly
when the expressed as an in vivo dosage of gene editing composition
packaged in a nanoparticle with or without functional molecules.
Dosages can be, for example 0.1 mg/kg to about 1,000 mg/kg, or 0.5
mg/kg to about 1,000 mg/kg, or 1 mg/kg to about 1,000 mg/kg, or
about 10 mg/kg to about 500 mg/kg, or about 20 mg/kg to about 500
mg/kg per dose, or 20 mg/kg to about 100 mg/kg per dose, or 25
mg/kg to about 75 mg/kg per dose, or about 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, or 75 mg/kg per dose.
In other embodiments, dosages are expressed in mg/ml, particularly
when the expressed as an ex vivo dosage of gene editing composition
packaged in a nanoparticle with or without functional molecules.
Dosages can be, for example 0.01 mg/ml to about 100 mg/ml, or about
0.5 mg/ml to about 50 mg/ml, or about 1 mg/ml to about 10 mg/ml per
dose to a cell population of 10.sup.6 cells.
As discussed above, gene editing technology can be administered
without, but is preferably administered with at least one donor
oligonucleotide. Such donors can be administered at similar dosages
as the gene editing technology. Compositions should include an
amount of donor fragment effective to recombine at the target site
in the presence of a gene editing technology such as triplex
forming molecules.
Potentiating Agents
The methods can include contacting cells with an effective amount
potentiating agents. Preferably the amount of potentiating agent is
effective to increase gene modification when used in combination
with a gene modifying technology, compared to using the gene
modifying technology in the absence of the potentiating agent.
Exemplary dosages for SCF include, about 0.01 mg/kg to about 250
mg/kg, or about 0.1 mg/kg to about 100 mg/kg, or about 0.5 mg/kg to
about 50 mg/kg, or about 0.75 mg/kg to about 10 mg/kg.
Dosages for CHK1 inhibitors are known in the art, and many of these
are in clinical trial. Accordingly, the dosage can be selected by
the practitioner based on known, preferred humans dosages. In
preferred embodiments, the dosage is below the
lowest-observed-adverse-effect level (LOAEL), and is preferably a
no observed adverse effect level (NOAEL) dosage.
1. Ex Vivo Gene Therapy
In some embodiments, ex vivo gene therapy of cells is used for the
treatment of a genetic disorder in a subject. For ex vivo gene
therapy, cells are isolated from a subject and contacted ex vivo
with the compositions to produce cells containing mutations in or
adjacent to genes. In a preferred embodiment, the cells are
isolated from the subject to be treated or from a syngenic host.
Target cells are removed from a subject prior to contacting with a
gene editing composition and preferably a potentiating factor. The
cells can be hematopoietic progenitor or stem cells. In a preferred
embodiment, the target cells are CD34.sup.+ hematopoietic stem
cells. Hematopoietic stem cells (HSCs), such as CD34.sup.+ cells
are multipotent stem cells that give rise to all the blood cell
types including erythrocytes. Therefore, CD34+ cells can be
isolated from a patient with, for example, thalassemia, sickle cell
disease, or a lysosomal storage disease, the mutant gene altered or
repaired ex-vivo using the disclosed compositions and methods, and
the cells reintroduced back into the patient as a treatment or a
cure.
Stem cells can be isolated and enriched by one of skill in the art.
Methods for such isolation and enrichment of CD34.sup.+ and other
cells are known in the art and disclosed for example in U.S. Pat.
Nos. 4,965,204; 4,714,680; 5,061,620; 5,643,741; 5,677,136;
5,716,827; 5,750,397 and 5,759,793. As used herein in the context
of compositions enriched in hematopoietic progenitor and stem
cells, "enriched" indicates a proportion of a desirable element
(e.g. hematopoietic progenitor and stem cells) which is higher than
that found in the natural source of the cells. A composition of
cells may be enriched over a natural source of the cells by at
least one order of magnitude, preferably two or three orders, and
more preferably 10, 100, 200 or 1000 orders of magnitude.
In humans, CD34.sup.+ cells can be recovered from cord blood, bone
marrow or from blood after cytokine mobilization effected by
injecting the donor with hematopoietic growth factors such as
granulocyte colony stimulating factor (G-CSF), granulocyte-monocyte
colony stimulating factor (GM-CSF), stem cell factor (SCF)
subcutaneously or intravenously in amounts sufficient to cause
movement of hematopoietic stem cells from the bone marrow space
into the peripheral circulation. Initially, bone marrow cells may
be obtained from any suitable source of bone marrow, e.g. tibiae,
femora, spine, and other bone cavities. For isolation of bone
marrow, an appropriate solution may be used to flush the bone,
which solution will be a balanced salt solution, conveniently
supplemented with fetal calf serum or other naturally occurring
factors, in conjunction with an acceptable buffer at low
concentration, generally from about 5 to 25 mM. Convenient buffers
include Hepes, phosphate buffers, lactate buffers, etc.
Cells can be selected by positive and negative selection
techniques. Cells can be selected using commercially available
antibodies which bind to hematopoietic progenitor or stem cell
surface antigens, e.g. CD34, using methods known to those of skill
in the art. For example, the antibodies may be conjugated to
magnetic beads and immunogenic procedures utilized to recover the
desired cell type. Other techniques involve the use of fluorescence
activated cell sorting (FACS). The CD34 antigen, which is found on
progenitor cells within the hematopoietic system of non-leukemic
individuals, is expressed on a population of cells recognized by
the monoclonal antibody My-10 (i.e., express the CD34 antigen) and
can be used to isolate stem cell for bone marrow transplantation.
My-10 deposited with the American Type Culture Collection
(Rockville, Md.) as HB-8483 is commercially available as anti-HPCA
1. Additionally, negative selection of differentiated and
"dedicated" cells from human bone marrow can be utilized, to select
against substantially any desired cell marker. For example,
progenitor or stem cells, most preferably CD34.sup.+ cells, can be
characterized as being any of CD3.sup.-, CDT, CD8.sup.-,
CD10.sup.-, CD14.sup.-, CD15.sup.-, CD19.sup.-, CD20.sup.-,
CD33.sup.-, Class II HLA.sup.+ and Thy-1.sup.+.
Once progenitor or stem cells have been isolated, they may be
propagated by growing in any suitable medium. For example,
progenitor or stem cells can be grown in conditioned medium from
stromal cells, such as those that can be obtained from bone marrow
or liver associated with the secretion of factors, or in medium
including cell surface factors supporting the proliferation of stem
cells. Stromal cells may be freed of hematopoietic cells employing
appropriate monoclonal antibodies for removal of the undesired
cells.
The isolated cells are contacted ex vivo with a combination of
triplex-forming molecules and donor oligonucleotides in amounts
effective to cause the desired mutations in or adjacent to genes in
need of repair or alteration, for example the human beta-globin or
.alpha.-L-iduronidase gene. These cells are referred to herein as
modified cells. Methods for transfection of cells with
oligonucleotides and peptide nucleic acids are well known in the
art (Koppelhus, et al., Adv. Drug Deliv. Rev., 55(2): 267-280
(2003)). It may be desirable to synchronize the cells in S-phase to
further increase the frequency of gene correction. Methods for
synchronizing cultured cells, for example, by double thymidine
block, are known in the art (Zielke, et al., Methods Cell Biol.,
8:107-121 (1974)).
The modified cells can be maintained or expanded in culture prior
to administration to a subject. Culture conditions are generally
known in the art depending on the cell type. Conditions for the
maintenance of CD34.sup.+ in particular have been well studied, and
several suitable methods are available. A common approach to ex
vivo multi-potential hematopoietic cell expansion is to culture
purified progenitor or stem cells in the presence of early-acting
cytokines such as interleukin-3. It has also been shown that
inclusion, in a nutritive medium for maintaining hematopoietic
progenitor cells ex vivo, of a combination of thrombopoietin (TPO),
stem cell factor (SCF), and flt3 ligand (Flt-3L; i.e., the ligand
of the flt3 gene product) was useful for expanding primitive (i.e.,
relatively non-differentiated) human hematopoietic progenitor cells
in vitro, and that those cells were capable of engraftment in
SCID-hu mice (Luens et al., 1998, Blood 91:1206-1215). In other
known methods, cells can be maintained ex vivo in a nutritive
medium (e.g., for minutes, hours, or 3, 6, 9, 13, or more days)
including murine prolactin-like protein E (mPLP-E) or murine
prolactin-like protein F (mPIP-F; collectively mPLP-E/IF) (U.S.
Pat. No. 6,261,841). It will be appreciated that other suitable
cell culture and expansion method can be used in accordance with
the invention as well. Cells can also be grown in serum-free
medium, as described in U.S. Pat. No. 5,945,337.
In another embodiment, the modified hematopoietic stem cells are
differentiated ex vivo into CD4.sup.+ cells culture using specific
combinations of interleukins and growth factors prior to
administration to a subject using methods well known in the art.
The cells may be expanded ex vivo in large numbers, preferably at
least a 5-fold, more preferably at least a 10-fold and even more
preferably at least a 20-fold expansion of cells compared to the
original population of isolated hematopoietic stem cells.
In another embodiment cells for ex vivo gene therapy, the cells to
be used can be dedifferentiated somatic cells. Somatic cells can be
reprogrammed to become pluripotent stem-like cells that can be
induced to become hematopoietic progenitor cells. The hematopoietic
progenitor cells can then be treated with triplex-forming molecules
and donor oligonucleotides as described above with respect to
CD34.sup.+ cells to produce recombinant cells having one or more
modified genes. Representative somatic cells that can be
reprogrammed include, but are not limited to fibroblasts,
adipocytes, and muscles cells. Hematopoietic progenitor cells from
induced stem-like cells have been successfully developed in the
mouse (Hanna, J. et al. Science, 318:1920-1923 (2007)).
To produce hematopoietic progenitor cells from induced stem-like
cells, somatic cells are harvested from a host. In a preferred
embodiment, the somatic cells are autologous fibroblasts. The cells
are cultured and transduced with vectors encoding Oct4, Sox2, Klf4,
and c-Myc transcription factors. The transduced cells are cultured
and screened for embryonic stem cell (ES) morphology and ES cell
markers including, but not limited to AP, SSEA1, and Nanog. The
transduced ES cells are cultured and induced to produce induced
stem-like cells. Cells are then screened for CD41 and c-kit markers
(early hematopoietic progenitor markers) as well as markers for
myeloid and erythroid differentiation.
The modified hematopoietic stem cells or modified induced
hematopoietic progenitor cells are then introduced into a subject.
Delivery of the cells may be effected using various methods and
includes most preferably intravenous administration by infusion as
well as direct depot injection into periosteal, bone marrow and/or
subcutaneous sites.
The subject receiving the modified cells may be treated for bone
marrow conditioning to enhance engraftment of the cells. The
recipient may be treated to enhance engraftment, using a radiation
or chemotherapeutic treatment prior to the administration of the
cells. Upon administration, the cells will generally require a
period of time to engraft. Achieving significant engraftment of
hematopoietic stem or progenitor cells typically takes weeks to
months.
A high percentage of engraftment of modified hematopoietic stem
cells is not envisioned to be necessary to achieve significant
prophylactic or therapeutic effect. It is expected that the
engrafted cells will expand over time following engraftment to
increase the percentage of modified cells. In some embodiments, the
modified cells have a corrected .alpha.-L-iduronidase gene.
Therefore, in a subject with Hurler syndrome, the modified cells
are expected to improve or cure the condition. It is expected that
engraftment of only a small number or small percentage of modified
hematopoietic stem cells will be required to provide a prophylactic
or therapeutic effect.
In preferred embodiments, the cells to be administered to a subject
will be autologous, e.g. derived from the subject, or syngenic.
2. In Vivo Gene Therapy
The disclosed compositions can be administered directly to a
subject for in vivo gene therapy.
a. Pharmaceutical Formulations
The disclosed compositions are preferably employed for therapeutic
uses in combination with a suitable pharmaceutical carrier. Such
compositions include an effective amount of the composition, and a
pharmaceutically acceptable carrier or excipient.
It is understood by one of ordinary skill in the art that
nucleotides administered in vivo are taken up and distributed to
cells and tissues (Huang, et al., FEBS Lett., 558(1-3):69-73
(2004)). For example, Nyce, et al. have shown that antisense
oligodeoxynucleotides (ODNs) when inhaled bind to endogenous
surfactant (a lipid produced by lung cells) and are taken up by
lung cells without a need for additional carrier lipids (Nyce, et
al., Nature, 385:721-725 (1997)). Small nucleic acids are readily
taken up into T24 bladder carcinoma tissue culture cells (Ma, et
al., Antisense Nucleic Acid Drug Dev., 8:415-426 (1998)).
The disclosed compositions including triplex-forming molecules,
such as TFOs and PNAs, and donor fragments may be in a formulation
for administration topically, locally or systemically in a suitable
pharmaceutical carrier. Remington's Pharmaceutical Sciences, 15th
Edition by E. W. Martin (Mark Publishing Company, 1975), discloses
typical carriers and methods of preparation. The compound may also
be encapsulated in suitable biocompatible microcapsules,
microparticles, nanoparticles, or microspheres formed of
biodegradable or non-biodegradable polymers or proteins or
liposomes for targeting to cells. Such systems are well known to
those skilled in the art and may be optimized for use with the
appropriate nucleic acid.
Various methods for nucleic acid delivery are described, for
example, in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1989); and
Ausubel, et al., Current Protocols in Molecular Biology, John Wiley
& Sons, New York (1994). Such nucleic acid delivery systems
include the desired nucleic acid, by way of example and not by
limitation, in either "naked" form as a "naked" nucleic acid, or
formulated in a vehicle suitable for delivery, such as in a complex
with a cationic molecule or a liposome forming lipid, or as a
component of a vector, or a component of a pharmaceutical
composition. The nucleic acid delivery system can be provided to
the cell either directly, such as by contacting it with the cell,
or indirectly, such as through the action of any biological
process. The nucleic acid delivery system can be provided to the
cell by endocytosis, receptor targeting, coupling with native or
synthetic cell membrane fragments, physical means such as
electroporation, combining the nucleic acid delivery system with a
polymeric carrier such as a controlled release film or nanoparticle
or microparticle, using a vector, injecting the nucleic acid
delivery system into a tissue or fluid surrounding the cell, simple
diffusion of the nucleic acid delivery system across the cell
membrane, or by any active or passive transport mechanism across
the cell membrane. Additionally, the nucleic acid delivery system
can be provided to the cell using techniques such as
antibody-related targeting and antibody-mediated immobilization of
a viral vector.
Formulations for topical administration may include ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and
powders. Conventional pharmaceutical carriers, aqueous, powder or
oily bases, or thickeners can be used as desired.
Formulations suitable for parenteral administration, such as, for
example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions, solutions or emulsions that can include suspending
agents, solubilizers, thickening agents, dispersing agents,
stabilizers, and preservatives. Formulations for injection may be
presented in unit dosage form, e.g., in ampules or in multi-dose
containers, optionally with an added preservative. The compositions
may take such forms as sterile aqueous or nonaqueous solutions,
suspensions and emulsions, which can be isotonic with the blood of
the subject in certain embodiments. Examples of nonaqueous solvents
are polypropylene glycol, polyethylene glycol, vegetable oil such
as olive oil, sesame oil, coconut oil, arachis oil, peanut oil,
mineral oil, injectable organic esters such as ethyl oleate, or
fixed oils including synthetic mono or di-glycerides. Aqueous
carriers include water, alcoholic/aqueous solutions, emulsions or
suspensions, including saline and buffered media. Parenteral
vehicles include sodium chloride solution, 1,3-butandiol, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's or fixed
oils. Intravenous vehicles include fluid and nutrient replenishers,
and electrolyte replenishers (such as those based on Ringer's
dextrose). Preservatives and other additives may also be present
such as, for example, antimicrobials, antioxidants, chelating
agents and inert gases. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil including synthetic mono- or
di-glycerides may be employed. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables. Carrier
formulation can be found in Remington's Pharmaceutical Sciences,
Mack Publishing Co., Easton, Pa. Those of skill in the art can
readily determine the various parameters for preparing and
formulating the compositions without resort to undue
experimentation.
The disclosed compositions alone or in combination with other
suitable components, can also be made into aerosol formulations
(i.e., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and air. For administration by inhalation, the compounds are
delivered in the form of an aerosol spray presentation from
pressurized packs or a nebulizer, with the use of a suitable
propellant.
In some embodiments, the compositions include pharmaceutically
acceptable carriers with formulation ingredients such as salts,
carriers, buffering agents, emulsifiers, diluents, excipients,
chelating agents, fillers, drying agents, antioxidants,
antimicrobials, preservatives, binding agents, bulking agents,
silicas, solubilizers, or stabilizers. In one embodiment, the
triplex-forming molecules and/or donor oligonucleotides are
conjugated to lipophilic groups like cholesterol and lauric and
lithocholic acid derivatives with C32 functionality to improve
cellular uptake. For example, cholesterol has been demonstrated to
enhance uptake and serum stability of siRNA in vitro (Lorenz, et
al., Bioorg. Med. Chem. Lett., 14(19):4975-4977 (2004)) and in vivo
(Soutschek, et al., Nature, 432(7014):173-178 (2004)). In addition,
it has been shown that binding of steroid conjugated
oligonucleotides to different lipoproteins in the bloodstream, such
as LDL, protect integrity and facilitate biodistribution (Rump, et
al., Biochem. Pharmacol., 59(11):1407-1416 (2000)). Other groups
that can be attached or conjugated to the compound described above
to increase cellular uptake, include acridine derivatives;
cross-linkers such as psoralen derivatives, azidophenacyl,
proflavin, and azidoproflavin; artificial endonucleases; metal
complexes such as EDTA-Fe(II) and porphyrin-Fe(II); alkylating
moieties; nucleases such as alkaline phosphatase; terminal
transferases; abzymes; cholesteryl moieties; lipophilic carriers;
peptide conjugates; long chain alcohols; phosphate esters;
radioactive markers; non-radioactive markers; carbohydrates; and
polylysine or other polyamines. U.S. Pat. No. 6,919,208 to Levy, et
al., also describes methods for enhanced delivery. These
pharmaceutical formulations may be manufactured in a manner that is
itself known, e.g., by means of conventional mixing, dissolving,
granulating, levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes.
b. Methods of Administration
In general, methods of administering compounds, including
oligonucleotides and related molecules, are well known in the art.
In particular, the routes of administration already in use for
nucleic acid therapeutics, along with formulations in current use,
provide preferred routes of administration and formulation for the
triplex-forming molecules described above. Preferably the
compositions are injected into the organism undergoing genetic
manipulation, such as an animal requiring gene therapy.
The disclosed compositions can be administered by a number of
routes including, but not limited to, oral, intravenous,
intraperitoneal, intramuscular, transdermal, subcutaneous, topical,
sublingual, rectal, intranasal, pulmonary, and other suitable
means. The compositions can also be administered via liposomes.
Such administration routes and appropriate formulations are
generally known to those of skill in the art.
Administration of the formulations may be accomplished by any
acceptable method which allows the gene editing compositions to
reach their targets.
Any acceptable method known to one of ordinary skill in the art may
be used to administer a formulation to the subject. The
administration may be localized (i.e., to a particular region,
physiological system, tissue, organ, or cell type) or systemic,
depending on the condition being treated.
Injections can be e.g., intravenous, intradermal, subcutaneous,
intramuscular, or intraperitoneal. In some embodiments, the
injections can be given at multiple locations. Implantation
includes inserting implantable drug delivery systems, e.g.,
microspheres, hydrogels, polymeric reservoirs, cholesterol
matrixes, polymeric systems, e.g., matrix erosion and/or diffusion
systems and non-polymeric systems, e.g., compressed, fused, or
partially-fused pellets. Inhalation includes administering the
composition with an aerosol in an inhaler, either alone or attached
to a carrier that can be absorbed. For systemic administration, it
may be preferred that the composition is encapsulated in
liposomes.
The compositions may be delivered in a manner which enables
tissue-specific uptake of the agent and/or nucleotide delivery
system. Techniques include using tissue or organ localizing
devices, such as wound dressings or transdermal delivery systems,
using invasive devices such as vascular or urinary catheters, and
using interventional devices such as stents having drug delivery
capability and configured as expansive devices or stent grafts.
The formulations may be delivered using a bioerodible implant by
way of diffusion or by degradation of the polymeric matrix. In
certain embodiments, the administration of the formulation may be
designed so as to result in sequential exposures to the
composition, over a certain time period, for example, hours, days,
weeks, months or years. This may be accomplished, for example, by
repeated administrations of a formulation or by a sustained or
controlled release delivery system in which the compositions are
delivered over a prolonged period without repeated administrations.
Administration of the formulations using such a delivery system may
be, for example, by oral dosage forms, bolus injections,
transdermal patches or subcutaneous implants. Maintaining a
substantially constant concentration of the composition may be
preferred in some cases.
Other delivery systems suitable include time-release, delayed
release, sustained release, or controlled release delivery systems.
Such systems may avoid repeated administrations in many cases,
increasing convenience to the subject and the physician. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include, for example, polymer-based
systems such as polylactic and/or polyglycolic acids,
polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides,
polyorthoesters, polyhydroxybutyric acid, and/or combinations of
these. Microcapsules of the foregoing polymers containing nucleic
acids are described in, for example, U.S. Pat. No. 5,075,109. Other
examples include non-polymer systems that are lipid-based including
sterols such as cholesterol, cholesterol esters, and fatty acids or
neutral fats such as mono-, di- and triglycerides; hydrogel release
systems; liposome-based systems; phospholipid based-systems;
silastic systems; peptide based systems; wax coatings; compressed
tablets using conventional binders and excipients; or partially
fused implants. Specific examples include erosional systems in
which the oligonucleotides are contained in a formulation within a
matrix (for example, as described in U.S. Pat. Nos. 4,452,775,
4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), or
diffusional systems in which an active component controls the
release rate (for example, as described in U.S. Pat. Nos.
3,832,253, 3,854,480, 5,133,974 and 5,407,686). The formulation may
be as, for example, microspheres, hydrogels, polymeric reservoirs,
cholesterol matrices, or polymeric systems. In some embodiments,
the system may allow sustained or controlled release of the
composition to occur, for example, through control of the diffusion
or erosion/degradation rate of the formulation containing the
triplex-forming molecules and donor oligonucleotides. In addition,
a pump-based hardware delivery system may be used to deliver one or
more embodiments.
Examples of systems in which release occurs in bursts include
systems in which the composition is entrapped in liposomes which
are encapsulated in a polymer matrix, the liposomes being sensitive
to specific stimuli, e.g., temperature, pH, light or a degrading
enzyme and systems in which the composition is encapsulated by an
ionically-coated microcapsule with a microcapsule core degrading
enzyme. Examples of systems in which release of the inhibitor is
gradual and continuous include, e.g., erosional systems in which
the composition is contained in a form within a matrix and
effusional systems in which the composition permeates at a
controlled rate, e.g., through a polymer. Such sustained release
systems can be in the form of pellets, or capsules.
Use of a long-term release implant may be particularly suitable in
some embodiments. "Long-term release," as used herein, means that
the implant containing the composition is constructed and arranged
to deliver therapeutically effective levels of the composition for
at least 30 or 45 days, and preferably at least 60 or 90 days, or
even longer in some cases. Long-term release implants are well
known to those of ordinary skill in the art, and include some of
the release systems described above.
c. Preferred Formulations for Mucosal and Pulmonary
Administration
Active agent(s) and compositions thereof can be formulated for
pulmonary or mucosal administration. The administration can include
delivery of the composition to the lungs, nasal, oral (sublingual,
buccal), vaginal, or rectal mucosa.
In one embodiment, the compounds are formulated for pulmonary
delivery, such as intranasal administration or oral inhalation. The
respiratory tract is the structure involved in the exchange of
gases between the atmosphere and the blood stream. The lungs are
branching structures ultimately ending with the alveoli where the
exchange of gases occurs. The alveolar surface area is the largest
in the respiratory system and is where drug absorption occurs. The
alveoli are covered by a thin epithelium without cilia or a mucus
blanket and secrete surfactant phospholipids. The respiratory tract
encompasses the upper airways, including the oropharynx and larynx,
followed by the lower airways, which include the trachea followed
by bifurcations into the bronchi and bronchioli. The upper and
lower airways are called the conducting airways. The terminal
bronchioli then divide into respiratory bronchiole, which then lead
to the ultimate respiratory zone, the alveoli, or deep lung. The
deep lung, or alveoli, is the primary target of inhaled therapeutic
aerosols for systemic drug delivery.
Pulmonary administration of therapeutic compositions comprised of
low molecular weight drugs has been observed, for example,
beta-androgenic antagonists to treat asthma. Other therapeutic
agents that are active in the lungs have been administered
systemically and targeted via pulmonary absorption. Nasal delivery
is considered to be a promising technique for administration of
therapeutics for the following reasons: the nose has a large
surface area available for drug absorption due to the coverage of
the epithelial surface by numerous microvilli, the subepithelial
layer is highly vascularized, the venous blood from the nose passes
directly into the systemic circulation and therefore avoids the
loss of drug by first-pass metabolism in the liver, it offers lower
doses, more rapid attainment of therapeutic blood levels, quicker
onset of pharmacological activity, fewer side effects, high total
blood flow per cm.sup.3, porous endothelial basement membrane, and
it is easily accessible.
The term aerosol as used herein refers to any preparation of a fine
mist of particles, which can be in solution or a suspension,
whether or not it is produced using a propellant. Aerosols can be
produced using standard techniques, such as ultrasonication or
high-pressure treatment.
Carriers for pulmonary formulations can be divided into those for
dry powder formulations and for administration as solutions.
Aerosols for the delivery of therapeutic agents to the respiratory
tract are known in the art. For administration via the upper
respiratory tract, the formulation can be formulated into a
solution, e.g., water or isotonic saline, buffered or un-buffered,
or as a suspension, for intranasal administration as drops or as a
spray. Preferably, such solutions or suspensions are isotonic
relative to nasal secretions and of about the same pH, ranging
e.g., from about pH 4.0 to about pH 7.4 or, from pH 6.0 to pH 7.0.
Buffers should be physiologically compatible and include, simply by
way of example, phosphate buffers. For example, a representative
nasal decongestant is described as being buffered to a pH of about
6.2. One skilled in the art can readily determine a suitable saline
content and pH for an innocuous aqueous solution for nasal and/or
upper respiratory administration.
Preferably, the aqueous solution is water, physiologically
acceptable aqueous solutions containing salts and/or buffers, such
as phosphate buffered saline (PBS), or any other aqueous solution
acceptable for administration to an animal or human. Such solutions
are well known to a person skilled in the art and include, but are
not limited to, distilled water, de-ionized water, pure or
ultrapure water, saline, phosphate-buffered saline (PBS). Other
suitable aqueous vehicles include, but are not limited to, Ringer's
solution and isotonic sodium chloride. Aqueous suspensions may
include suspending agents such as cellulose derivatives, sodium
alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting
agent such as lecithin. Suitable preservatives for aqueous
suspensions include ethyl and n-propyl p-hydroxybenzoate.
In another embodiment, solvents that are low toxicity organic (i.e.
nonaqueous) class 3 residual solvents, such as ethanol, acetone,
ethyl acetate, tetrahydrofuran, ethyl ether, and propanol may be
used for the formulations. The solvent is selected based on its
ability to readily aerosolize the formulation. The solvent should
not detrimentally react with the compounds. An appropriate solvent
should be used that dissolves the compounds or forms a suspension
of the compounds. The solvent should be sufficiently volatile to
enable formation of an aerosol of the solution or suspension.
Additional solvents or aerosolizing agents, such as freons, can be
added as desired to increase the volatility of the solution or
suspension.
In one embodiment, compositions may contain minor amounts of
polymers, surfactants, or other excipients well known to those of
the art. In this context, "minor amounts" means no excipients are
present that might affect or mediate uptake of the compounds in the
lungs and that the excipients that are present are present in
amount that do not adversely affect uptake of compounds in the
lungs.
Dry lipid powders can be directly dispersed in ethanol because of
their hydrophobic character. For lipids stored in organic solvents
such as chloroform, the desired quantity of solution is placed in a
vial, and the chloroform is evaporated under a stream of nitrogen
to form a dry thin film on the surface of a glass vial. The film
swells easily when reconstituted with ethanol. To fully disperse
the lipid molecules in the organic solvent, the suspension is
sonicated. Nonaqueous suspensions of lipids can also be prepared in
absolute ethanol using a reusable PARI LC Jet+ nebulizer (PARI
Respiratory Equipment, Monterey, Calif.).
C. Diseases to be Treated
Gene therapy is apparent when studied in the context of human
genetic diseases, for example, cystic fibrosis, hemophilia,
globinopathies such as sickle cell anemia and beta-thalassemia,
xeroderma pigmentosum, and lysosomal storage diseases, though the
strategies are also useful for treating non-genetic disease such as
HIV, in the context of ex vivo-based cell modification and also for
in vivo cell modification. The disclosed compositions are
especially useful to treat genetic deficiencies, disorders and
diseases caused by mutations in single genes, for example, to
correct genetic deficiencies, disorders and diseases caused by
point mutations. If the target gene contains a mutation that is the
cause of a genetic disorder, then the disclosed compositions can be
used for mutagenic repair that may restore the DNA sequence of the
target gene to normal. The target sequence can be within the coding
DNA sequence of the gene or within an intron. The target sequence
can also be within DNA sequences that regulate expression of the
target gene, including promoter or enhancer sequences.
If the target gene is an oncogene causing unregulated
proliferation, such as in a cancer cell, then the oligonucleotide
is useful for causing a mutation that inactivates the gene and
terminates or reduces the uncontrolled proliferation of the cell.
The oligonucleotide is also a useful anti-cancer agent for
activating a repressor gene that has lost its ability to repress
proliferation. The target gene can also be a gene that encodes an
immune regulatory factor, such as PD-1, in order to enhance the
host's immune response to a cancer.
Programmed cell death protein 1, also known as PD-1 and CD279
(cluster of differentiation 279), is a protein encoded by the PDCD1
gene. PD-1 has two ligands: PD-L1 and PD-L2. PD-1 is expressed on a
subset of thymocytes and up-regulated on T, B, and myeloid cells
after activation (Agata, et al., Int. Immunol., 8:765-772 (1996)).
PD-1 acts to antagonize signal transduction downstream of the TCR
after it binds a peptide antigen presented by the major
histocompatibility complex (MHC). It can function as an immune
checkpoint, by preventing the activation of T-cells, which in turn
reduces autoimmunity and promotes self-tolerance, but can also
reduce the body's ability to combat cancer. The inhibitory effect
of PD-1 to act through twofold mechanism of promoting apoptosis
(programmed cell death) in antigen specific T-cells in lymph nodes
while simultaneously reducing apoptosis in regulatory T cells
(suppressor T cells). Compositions that block PD-1, the PD-1
inhibitors, activate the immune system to attack tumors and are
therefore used with varying success to treat some types of
cancer.
Therefore, in some embodiments, compositions are used to treat
cancer. The gene modification technology can be designed to reduce
or prevent expression of PD-1, and administered in an effective
amount to do so.
The compositions can be used as antiviral agents, for example, when
designed to modify a specific a portion of a viral genome necessary
for proper proliferation or function of the virus.
Variants, Substitutions, and Exemplary PNAs
Preferred diseases and sequences of exemplary targeting sites,
triplex forming molecules, and donor oligonucleotides are discussed
in more detail below. Any of the sequences can also be modified as
disclosed herein or otherwise known in the art. For example, in
some embodiments, any of the triplex-forming sequences herein can
have one or more mutations (e.g., substitutions, deletions, or
insertions), such that the triplex-forming molecules still bind to
the target sequence.
Any of the triplex-forming sequences herein can be manufactured
using canonical nucleic acids or other suitable substitutes
including those disclosed herein (e.g., PNAs), without or without
any of the base, sugar, or backbone modifications discussed herein
or in WO 1996/040271, WO/2010/123983, and U.S. Pat. No.
8,658,608.
Any of the triplex-forming sequences herein can be peptide nucleic
acids. In some embodiments, one or more of the cytosines of any of
triplex-forming sequences herein is substituted with a
pseudoisocytosine. In some embodiments, all of the cytosines in the
Hoogsteen-binding portion of a triplex forming molecule are
substituted with pseudoisocytosine. In some embodiments, any of the
triplex-forming sequences herein, includes one or more of peptide
nucleic acid monomers substituted with a .gamma.PNA. In some
embodiments all of the peptide nucleic acid monomers in the
Hoogsteen-binding portion only, the Watson-Crick-binding portion
only, or across the entire PNA are substituted with .gamma.PNA
monomers. In particular embodiments, alternating residues are PNA
and .gamma.PNA in the Hoogsteen-binding portion only, the
Watson-Crick-binding portion only, or across the entire PNA are
substituted. In some embodiments, the .gamma.PNAs are miniPEG
.gamma.PNA, methyl .gamma.PNA, another .gamma. substitution
discussed above. In some embodiments, the PNA oligomer includes two
or more different .gamma.PNAs.
For example, in some embodiments, (1) some or all of the residues
in the Watson-Crick binding portion are .gamma.PNA (e.g.,
miniPEG-containing .gamma.PNA); (2) some or all of the residues in
the Hoogsteen binding portion are .gamma.PNA (e.g.,
miniPEG-containing .gamma.PNA); or (3) some or all of the residue
(in the Watson-Crick and/or Hoogsteen binding portions) are
.gamma.PNA (e.g., miniPEG-containing .gamma.PNA). Therefore, in
some embodiments any of the triplex foiming nucleic acid sequence
herein is a peptide nucleic acid wherein (1) all of the residues in
the Watson-Crick binding portion are .gamma.PNA (e.g.,
miniPEG-containing .gamma.PNA) and none of the residues is in
Hoogsteen binding portion are .gamma.PNA (e.g., miniPEG-containing
.gamma.PNA); (2) all of the residues in the Hoogsteen binding
portion are .gamma.PNA (e.g., miniPEG-containing .gamma.PNA) and
none of the residues is in Watson-Crick binding portion are
.gamma.PNA (e.g., miniPEG-containing .gamma.PNA); or (3) all of the
residues (in the Watson-Crick and Hoogsteen binding portions) are
.gamma.PNA (e.g., miniPEG-containing .gamma.PNA).
Preferred triplex molecules are bis-peptide nucleic acids with
pseudoisocytosine substituted for one or more cytosines,
particularly in the Hoogsteen-binding portion, and wherein some or
all of the PNA are .gamma.PNA.
Any of the triplex-forming sequences herein can have one or more
G-clamp monomers. For example, one or more cytosines or variant
thereof such as pseudoisocytosine in any of the triplex-forming
sequences herein can be substituted or otherwise modified to be a
clamp-G (9-(2-guanidinoethoxy) phenoxazine).
Any of the triplex-forming sequences herein can include a flexible
linker, linking, for example, a Hoogsteen-binding domain and a
Watson-Crick binding domain to form a bis-PNA. The sequences can be
linked with a flexible linker. For example, in some embodiments the
flexible linker includes about 1-10, more preferably 2-5, most
preferably about 3 units such as 8-amino-2,6,10-trioxaoctanoic acid
residues. Some molecules include N-terminal or C-terminal
non-binding residues, preferably positively charged. For example,
some molecules include 1-10, preferable 2-5, most preferably about
3 lysines at the N-terminus, the C-terminus, or a combination
thereof of the PNA.
For the disclosed sequences, "J" is pseudoisocyto sine, "O" is
flexible 8-amino-3,6-dioxaoctanoic acid, 6-aminohexanoic acid
monomers, "K" and "lys" are lysine. PNA sequences are generally
presented in an H-"nucleic acid sequence"-NH.sub.2 orientation. For
bis-PNA the Hoosten-binding portion is typically oriented up stream
(e.g., at the "H" end) of the linker, while the
Watson-Crick-binding portion is typically oriented downstream
(e.g., at the NH.sub.2 end) of the linker. Any of the donors can
include optional phosphorothioate internucleoside linkages,
particular between the three or four terminal 5' and three or four
terminal 3' nucleotides. Thus, each of the donor oligonucleotide
sequences disclosed herein is expressly disclosed without any
phosphorothioate internucleoside linkages, and with
phosphorothioate internucleoside linkages, preferably between the
three or four terminal 5' and three or four terminal 3'
nucleotides.
1. Globinopathies
Worldwide, globinopathies account for significant morbidity and
mortality. Over 1,200 different known genetic mutations affect the
DNA sequence of the human alpha-like (HBZ, HBA2, HBA1, and HBQ1)
and beta-like (HBE1, HBG1, HBD, and HBB) globin genes. Two of the
more prevalent and well-studied globinopathies are sickle cell
anemia and .beta.-thalassemia. Substitution of valine for glutamic
acid at position 6 of the .beta.-globin chain in patients with
sickle cell anemia predisposes to hemoglobin polymerization,
leading to sickle cell rigidity and vasoocclusion with resulting
tissue and organ damage. In patients with .beta.-thalassemia, a
variety of mutational mechanisms results in reduced synthesis of
.beta.-globin leading to accumulation of aggregates of unpaired,
insoluble .alpha.-chains that cause ineffective erythropoiesis,
accelerated red cell destruction, and severe anemia.
Together, globinopathies represent the most common single-gene
disorders in man. Triplex forming oligonucleotides are particularly
well suited to treat globinopathies, as they are single gene
disorders caused by point mutations. Triplex forming molecules
disclosed herein are effective at binding to the human
.beta.-globin both in vitro and in living cells, both ex vivo and
in vivo in animals. Experimental results also demonstrate
correction of a thalassemia-associated mutation in vivo in a
transgenic mouse carrying a human beta globin gene with the
IVS2-654 thalassemia mutation (in place of the endogenous mouse
beta globin) with correction of the mutation in 4% of the total
bone marrow cells, cure of the anemia with blood hemoglobin levels
showing a sustained elevation into the normal range, reversal of
extramedullary hematopoiesis and reversal of splenomegaly, and
reduction in reticulocyte counts, following systemic administration
of PNA and DNA containing nanoparticles.
.beta.-thalassemia is an unstable hemoglobinopathy leading to the
precipitation of .alpha.-hemoglobin within RBCs resulting in a
severe hemolytic anemia. Patients experience jaundice and
splenomegaly, with substantially decreased blood hemoglobin
concentrations necessitating repeated transfusions, typically
resulting in severe iron overload with time. Cardiac failure due to
myocardial siderosis is a major cause of death from
.beta.-thalassemia by the end of the third decade. Reduction of
repeated blood transfusions in these patients is therefore of
primary importance to improve patient outcomes.
a. Exemplary .beta.-Globin Gene Target Sites
In the .beta.-globin gene sequence, particularly in the introns,
there are many good third-strand binding sites that may be utilized
in the methods disclosed herein. A portion of the GenBank sequence
of the chromosome-11 human-native hemoglobin-gene cluster (GenBank:
U01317.1--Human beta globin region on chromosome 11--LOCUS HUMHBB,
73308 bp ds-DNA) from base 60001 to base 66060 is presented below.
The start of the gene coding sequence at position 62187-62189 (or
positions 2187-2189 of SEQ ID NO:13) is indicated by wave
underlining. This portion of the GenBank sequence contains the
native .beta. globin gene sequence. In sickle cell hemoglobin the
adenine base at position 62206 (or position 2206 as listed in SEQ
ID NO:13, indicated in bold and heavy underlining) is mutated to a
thymine. Other common point mutations occur in intron 2 (IVS2),
which is highlighted in the sequence below by italics (SEQ ID
NO:14) and corresponds with nucleotides 2,632-3,481 of SEQ ID
NO:13. Mutations include IVS2-1, IVS2-566, IVS2-654, IVS2-705, and
IVS2-745, which are also shown in bold and heavy underlining;
numbering relative to the start of intron 2.
Exemplary triplex forming molecule binding sites, are provided in,
for example, WO 1996/040271, WO/2010/123983, and U.S. Pat. No.
8,658,608, and in the working Examples below. Target regions can be
reference based on the coding strand of genomic DNA, or the
complementary non-coding sequence thereto (e.g., the Watson or
Crick stand). Exemplary target regions are identified with
reference to the coding sequence of the globin gene sequence in the
sequence below by double underlining and a combination of
underlining and double underlining (wherein the underlining is
optional additional binding sequence). Additionally, for each
targeting sequence identified, the complementary target sequence on
the reverse non-coding strand is also explicitly disclosed as a
triplex forming molecule binding sequence.
Accordingly, triplex forming molecules can be designed to bind a
target region on either the coding or non-coding strand. However,
as discussed above, triplex-forming molecules, such as PNA and
tcPNA preferably invade the target duplex, displacement of the
polypyrimidine, and induce triplex formation with the displaced
polypurine.
TABLE-US-00007 (SEQ ID NO: 13 - full sequence; SEQ ID NO: 14 -
sequence in italics).
AAAGCTCTTGCTTTGACAATTTTGGTCTTTCAGAATACTATAAATATAACCTATATTATA
ATTTCATAAAGTCTGTGCATTTTCTTTGACCCAGGATATTTGCAAAAGACATATTCAAAC
TTCCGCAGAACACTTTATTTCACATATACATGCCTCTTATATCAGGGATGTGAAACAGGG
TCTTGAAAACTGTCTAAATCTAAAACAATGCTAATGCAGGTTTAAATTTAATAAAATAAA
ATCCAAAATCTAACAGCCAAGTCAAATCTGTATGTTTTAACATTTAAAATATTTTAAAGA
CGTCTTTTCCCAGGATTCAACATGTGAAATCTTTTCTCAGGGATACACGTGTGCCTAGAT
CCTCATTGCTTTAGTTTTTTACAGAGGAATGAATATAAAAAGAAAATACTTAAATTTTAT
CCCTCTTACCTCTATAATCATACATAGGCATAATTTTTTAACCTAGGCTCCAGATAGCCA
TAGAAGAACCAAACACTTTCTGCGTGTGTGAGAATAATCAGAGTGAGATTTTTTCACAAG
TACCTGATGAGGGTTGAGACAGGTAGAAAAAGTGAGAGATCTCTATTTATTTAGCAATAA
TAGAGAAAGCATTTAAGAGAATAAAGCAATGGAAATAAGAAATTTGTAAATTTCCTTCTG
ATAACTAGAAATAGAGGATCCAGTTTCTTTTGGTTAACCTAAATTTTATTTCATTTTATT
GTTTTATTTTATTTTATTTTATTTTATTTTGTGTAATCGTAGTTTCAGAGTGTTAGAGCT
GAAAGGAAGAAGTAGGAGAAACATGCAAAGTAAAAGTATAACACTTTCCTTACTAAACCG
ACTGGGTTTCCAGGTAGGGGCAGGATTCAGGATGACTGACAGGGCCCTTAGGGAACACTG
AGACCCTACGCTGACCTCATAAATGCTTGCTACCTTTGCTGTTTTAATTACATCTTTTAA
TAGCAGGAAGCAGAACTCTGCACTTCAAAAGTTTTTCCTCACCTGAGGAGTTAATTTAGT
ACAAGGGGAAAAAGTACAGGGGGATGGGAGAAAGGCGATCACGTTGGGAAGCTATAGAGA
AAGAAGAGTAAATTTTAGTAAAGGAGGTTTAAACAAACAAAATATAAAGAGAAATAGGAA
CTTGAATCAAGGAAATGATTTTAAAACGCAGTATTCTTAGTGGACTAGAGGAAAAAAATA
ATCTGAGCCAAGTAGAAGACCTTTTCCCCTCCTACCCCTACTTTCTAAGTCACAGAGGCT
TTTTGTTCCCCCAGACACTCTTGCAGATTAGTCCAGGCAGAAACAGTTAGATGTCCCCAG
TTAACCTCCTATTTGACACCACTGATTACCCCATTGATAGTCACACTTTGGGTTGTAAGT
GACTTTTTATTTATTTGTATTTTTGACTGCATTAAGAGGTCTCTAGTTTTTTATCTCTTG
TTTCCCAAAACCTAATAAGTAACTAATGCACAGAGCACATTGATTTGTATTTATTCTATT
TTTAGACATAATTTATTAGCATGCATGAGCAAATTAAGAAAAACAACAACAAATGAATGC
ATATATATGTATATGTATGTGTGTATATATACACATATATATATATATTTTTTTTCTTTT
CTTACCAGAAGGTTTTAATCCAAATAAGGAGAAGATATGCTTAGAACTGAGGTAGAGTTT
TCATCCATTCTGTCCTGTAAGTATTTTGCATATTCTGGAGACGCAGGAAGAGATCCATCT
ACATATCCCAAAGCTGAATTATGGTAGACAAAGCTCTTCCACTTTTAGTGCATCAATTTC
TTATTTGTGTAATAAGAAAATTGGGAAAACGATCTTCAATATGCTTACCAAGCTGTGATT
CCAAATATTACGTAAATACACTTGCAAAGGAGGATGTTTTTAGTAGCAATTTGTACTGAT
GGTATGGGGCCAAGAGATATATCTTAGAGGGAGGGCTGAGGGTTTGAAGTCCAACTCCTA
AGCCAGTGCCAGAAGAGCCAAGGACAGGTACGGCTGTCATCACTTAGACCTCACCCTGTG
GAGCCACACCCTAGGGTTGGCCAATCTACTCCCAGGAGCAGGGAGGGCAGGAGCCAGGGC
TGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTG
##STR00003##
TTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGGTGGTGAGGCCCTGGGCAGGT
TGGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCATGTGGAGACA
GAGAAGACTCTTGGGTTTCTGATAGGCACTGACTCTCTCTGCCTATTGGTCTATTTTCCC
ACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGAT
CTGTCCACTCCTGATGCTGTTATGGGCAACCCTAAGGTGAAGGCTCATGGCAAGAAAGTG
CTCGGTGCCTTTAGTGATGGCCTGGCTCACCTGGACAACCTCAAGGGCACCTTTGCCACA
##STR00004##
AAGTCTCAGGATCGTTTTAGTTTCTTTTATTTGCTGTTCATAACAATTGTTTTCTTTTGT
##STR00005##
AACATTGTGTATAACAAAAGGAAATATCTCTGAGATACATTAAGTAACTTAAAAAAAAAC
TTTACACAGTCTGCCTAGTACATTACTATTTGGAATATATGTGTGCTTATTTGCATATTC
ATAATCTCCCTACTTTATTTTCTTTTATTTTTAATTGATACATAATCATTATACATATTT
ATGGGTTAAAGTGTAATGTTTTAATATGTGTACACATATTGACCAAATCAGGGTAATTTT
##STR00006##
GCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTCACCCC
ACCAGTGCAGGCTGCCTATCAGAAAGTGGTGGCTGGTGTGGCTAATGCCCTGGCCCACAA
GTATCACTAAGCTCGCTTTCTTGCTGTCCAATTTCTATTAAAGGTTCCTTTGTTCCCTAA
GTCCAACTACTAAACTGGGGGATATTATGAAGGGCCTTGAGCATCTGGATTCTGCCTAAT
AAAAAACATTTATTTTCATTGCAATGATGTATTTAAATTATTTCTGAATATTTTACTAAA
AAGGGAATGTGGGAGGTCAGTGCATTTAAAACATAAAGAAATGAAGAGCTAGTTCAAACC
TTGGGAAAATACACTATATCTTAAACTCCATGAAAGAAGGTGAGGCTGCAAACAGCTAAT
GCACATTGGCAACAGCCCTGATGCCTATGCCTTATTCATCCCTCAGAAAAGGATTCAAGT
AGAGGCTTGATTTGGAGGTTAAAGTTTTGCTATGCTGTATTTTACATTACTTATTGTTTT
AGCTGTCCTCATGAATGTCTTTTCACTACCCATTTGCTTATCCTGCATCTCTCAGCCTTG
ACTCCACTCAGTTCTCTTGCTTAGAGATACCACCTTTCCCCTGAAGTGTTCCTTCCATGT
TTTACGGCGAGATGGTTTCTCCTCGCCTGGCCACTCAGCCTTAGTTGTCTCTGTTGTCTT
ATAGAGGTCTACTTGAAGAAGGAAAAACAGGGGGCATGGTTTGACTGTCCTGTGAGCCCT
TCTTCCCTGCCTCCCCCACTCACAGTGACCCGGAATCTGCAGTGCTAGTCTCCCGGAACT
ATCACTCTTTCACAGTCTGCTTTGGAAGGACTGGGCTTAGTATGAAAAGTTAGGACTGAG
AAGAATTTGAAAGGGGGCTTTTTGTAGCTTGATATTCACTACTGTCTTATTACCCTATCA
TAGGCCCACCCCAAATGGAAGTCCCATTCTTCCTCAGGATGTTTAAGATTAGCATTCAGG
AAGAGATCAGAGGTCTGCTGGCTCCCTTATCATGTCCCTTATGGTGCTTCTGGCTCTGCA
GTTATTAGCATAGTGTTACCATCAACCACCTTAACTTCATTTTTCTTATTCAATACCTAG
GTAGGTAGATGCTAGATTCTGGAAATAAAATATGAGTCTCAAGTGGTCCTTGTCCTCTCT
CCCAGTCAAATTCTGAATCTAGTTGGCAAGATTCTGAAATCAAGGCATATAATCAGTAAT
AAGTGATGATAGAAGGGTATATAGAAGAATTTTATTATATGAGAGGGTGAAACCTAAAAT
GAAATGAAATCAGACCCTTGTCTTACACCATAAACAAAAATAAATTTGAATGGGTTAAAG
AATTAAACTAAGACCTAAAACCATAAAAATTTTTAAAGAAATCAAAAGAAGAAAATTCTA
ATATTCATGTTGCAGCCGTTTTTTGAATTTGATATGAGAAGCAAAGGCAACAAAAGGAAA
AATAAAGAAGTGAGGCTACATCAAACTAAAAAATTTCCACACAAAAAAGAAAACAATGAA
CAAATGAAAGGTGAACCATGAAATGGCATATTTGCAAACCAAATATTTCTTAAATATTTT
GGTTAATATCCAAAATATATAAGAAACACAGATGATTCAATAACAAACAAAAAATTAAAA
ATAGGAAAATAAAAAAATTAAAAAGAAGAAAATCCTGCCATTTATGCGAGAATTGATGAA
CCTGGAGGATGTAAAACTAAGAAAAATAAGCCTGACACAAAAAGACAAATACTACACAAC
CTTGCTCATATGTGAAACATAAAAAAGTCACTCTCATGGAAACAGACAGTAGAGGTATGG
TTTCCAGGGGTTGGGGGTGGGAGAATCAGGAAACTATTACTCAAAGGGTATAAAATTTCA
GTTATGTGGGATGAATAAATTCTAGATATCTAATGTACAGCATCGTGACTGTAGTTAATT
GTACTGTAAGTATATTTAAAATTTGCAAAGAGAGTAGATTTTTTTGTTTTTTTAGATGGA
GTTTTGCTCTTGTTGTCCAGGCTGGAGTGCAATGGCAAGATCTTGGCTCACTGCAACCTC
CGCCTCCTGGGTTCAAGCAAATCTCCTGCCTCAGCCTCCCGAGTAGCTGGGATTACAGGC
ATGCGACACCATGCCCAGCTAATTTTGTATTTTTAGTAGAGACGGGGTTTCTCCATGTTG
GTCAGGCTGATCCGCCTCCTCGGCCACCAAAGGGCTGGGATTACAGGCGTGACCACCGGG
CCTGGCCGAGAGTAGATCTTAAAAGCATTTACCACAAGAAAAAGGTAACTATGTGAGATA
ATGGGTATGTTAATTAGCTTGATTGTGGTAATCATTTCACAAGGTATACATATATTAAAA
CATCATGTTGTACACCTTAAATATATACAATTTTTATTTGTGAATGATACCTCAATAAAG
TTGAAGAATAATAAAAAAGAATAGACATCACATGAATTAAAAAACTAAAAAATAAAAAAA
TGCATCTTGATGATTAGAATTGCATTCTTGATTTTTCAGATACAAATATCCATTTGACTG
b. Exemplary Triplex Forming Sequences
i. Beta Thalassemia
Gene editing molecules can be designed based on the guidance
provided herein and otherwise known in the art. Exemplary triplex
forming molecule and donor sequences, are provided in, for example,
WO 1996/040271, WO/2010/123983, and U.S. Pat. No. 8,658,608, and in
the working Examples below, and can be altered to include one or
more of the modifications disclosed herein.
Triplex forming molecules can include a sequence substantially
complementary to the polypurine strand of the
polypyrimidine:polypurine target motif. In some embodiments, the
triplex forming molecules target a region corresponding to
nucleotides 566-577, optionally 566-583 or more of SEQ ID NO:14; a
region corresponding to nucleotides 807-813, optionally 807-824 or
more of SEQ ID NO:14; or a region corresponding to nucleotides
605-611, optionally 605-621 of SEQ ID NO:14. Therefore in some
embodiments, the triplex-forming molecules can form a
triple-stranded molecule with the sequence including GAAAGAAAGAGA
(SEQ ID NO:15) or TGCCCTGAAAGAAAGAGA (SEQ ID NO:16) or GGAGAAA (SEQ
ID NO:17) or AGAATGGTGCAAAGAGG (SEQ ID NO:18) or AAAAGGG (SEQ ID
NO:19) or ACATGATTAGCAAAAGGG (SEQ ID NO:20).
Accordingly, in some embodiments, the triplex-folining molecule
includes the nucleic acid sequence CTTTCTTTCTCT (SEQ ID NO:21),
preferable includes the sequence CTTTCTTTCTCT (SEQ ID NO:21) linked
to the sequence TCTCTTTCTTTC (SEQ ID NO:22), or more preferable
includes the sequence CTTTCTTTCTCT (SEQ ID NO:21) linked to the
sequence TCTCTTTCTTTCAGGGCA (SEQ ID NO:23).
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence TTTCCC (SEQ ID NO:24), preferable includes
the sequence TTTCCC (SEQ ID NO:24) linked to the sequence CCCTTTT
(SEQ ID NO:25), or more preferable includes the sequence TTTCCC
(SEQ ID NO:24) linked to the sequence CCCTTTTGCTAATCATGT (SEQ ID
NO:26).
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence TTTCTCC (SEQ ID NO:27), preferable includes
the sequence TTTCTCC (SEQ ID NO:27) linked to the sequence CCTCTTT
(SEQ ID NO:28), or more preferable includes the sequence TTTCTCC
(SEQ ID NO:27) linked to the sequence CCTCTTTGCACCATTCT (SEQ ID
NO:29).
In some preferred embodiments, the triplex forming nucleic acid is
a peptide nucleic acid including the sequence JTTTJTTTJTJT (SEQ ID
NO:30) linked to the sequence TCTCTTTCTTTC (SEQ ID NO:22) or
TCTCTTTCTTTCAGGGCA (SEQ ID NO:23); or
a peptide nucleic acid including the sequence TTTTJJJ (SEQ ID
NO:31) linked to the sequence CCCTTTT (SEQ ID NO:25) or
CCCTTTTGCTAATCATGT (SEQ ID NO:26);
or a peptide nucleic acid including the sequence TTTJTJJ (SEQ ID
NO:32) linked to the sequence CCTCTTT (SEQ ID NO:28) or
CCTCTTTGCACCATTCT (SEQ ID NO:29),
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments, the triplex forming molecule is a peptide
nucleic acid including the sequence
lys-lys-lys-JTTTJTTTJTJT-OOO-TCTCTTTCTTTCAGGGCA-lys-lys-lys (SEQ ID
NO:33), or
lys-lys-lys-TTTTJJJ-OOO-CCCTTTTGCTAATCATGT-lys-lys-lys (SEQ ID
NO:34), or
lys-lys-lys-TTTJTJJ-OOO-CCTCTTTGCACCATTCT-lys-lys-lys (SEQ ID
NO:35);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA. In even more specific embodiments, the bolded and
underlined residues are miniPEG-containing .gamma.PNA.
In other embodiments, the triplex forming nucleic acid is a peptide
nucleic acid including the sequence TJTTTTJTTJ (SEQ ID NO:36)
linked to the sequence CTTCTTTTCT (SEQ ID NO:37); or
TTJTTJTTTJ (SEQ ID NO:38) linked to the sequence CTTTCTTCTT (SEQ ID
NO:39); or
JJJTJJTTJT (SEQ ID NO:40) linked to the sequence TCTTCCTCCC (SEQ ID
NO:41); or
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments, the triplex forming nucleic acid is a
peptide nucleic acid including the sequence
lys-lys-lys-TJTTTTJTTJ-OOO-CTTCTTTTCT-lys-lys-lys (SEQ ID NO:42)
(IVS2-24); or
lys-lys-lys-TTJTTJTTTJ-OOO-CTTTCTTCTT-lys-lys-lys (SEQ ID NO:43)
(IVS2-512); or
lys-lys-lys-JJJTJJTTJT-OOO-TCTTCCTCCC-lys-lys-lys (SEQ ID NO:44)
(IVS2-830);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA. In even more specific embodiments, the bolded and
underlined residues are miniPEG-containing .gamma.PNA.
ii. Sickle Cell Disease
Preferred sequences that target the sickle cell disease mutation
(20) in the beta globin gene are also provided (see, e.g., FIG. 6).
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence CCTCTTC (SEQ ID NO:45), preferable includes
the sequence CCTCTTC (SEQ ID NO:45) linked to the sequence CTTCTCC
(SEQ ID NO:46), or more preferable includes the sequence CCTCTTC
(SEQ ID NO:45) linked to the sequence CTTCTCCAAAGGAGT (SEQ ID
NO:47) or CTTCTCCACAGGAGTCAG (SEQ ID NO:48) or
CTTCTCCACAGGAGTCAGGTGC (SEQ ID NO:205).
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence TTCCTCT (SEQ ID NO:49), preferable includes
the sequence TTCCTCT (SEQ ID NO:49) linked to the sequence TCTCCTT
(SEQ ID NO:50), or more preferable includes the sequence TTCCTCT
(SEQ ID NO:49) linked to the sequence TCTCCTTAAACCTGT (SEQ ID
NO:51) or TCTCCTTAAACCTGTCTT (SEQ ID NO:212).
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence TCTCTTCT (SEQ ID NO:52), preferable includes
the sequence TCTCTTCT (SEQ ID NO:52) linked to the sequence
TCTTCTCT (SEQ ID NO:53), or more preferable includes the sequence
TCTCTTCT (SEQ ID NO:52) linked to the sequence TCTTCTCTGTCTCCAC
(SEQ ID NO:54) or TCTTCTCTGTCTCCACAT (SEQ ID NO:55).
In some preferred embodiments for correction of Sickle Cell Disease
Mutation (e.g., FIG. 6), the triplex forming nucleic acid is a
peptide nucleic acid including the sequence JJTJTTJ (SEQ ID NO:56)
linked to the sequence CTTCTCC (SEQ ID NO:46) or CTTCTCCAAAGGAGT
(SEQ ID NO:47) or CTTCTCCACAGGAGTCAG (SEQ ID NO:48) or
CTTCTCCACAGGAGTCAGGTGC (SEQ ID NO:205);
or a peptide nucleic acid including the sequence TTJJTJT (SEQ ID
NO:214) linked to the sequence TCTCCTT (SEQ ID NO:50) or
TCTCCTTAAACCTGT (SEQ ID NO:51) or TCTCCTTAAACCTGTCTT (SEQ ID
NO:212);
or a peptide nucleic acid including the sequence TJTJTTJT (SEQ ID
NO:215) linked to the sequence TCTTCTCT (SEQ ID NO:53) or
TCTTCTCTGTCTCCAC (SEQ ID NO:54) or TCTTCTCTGTCTCCACAT (SEQ ID
NO:55);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments for correction of Sickle Cell Disease
Mutation (e.g., FIG. 6), the triplex forming nucleic acid is a
peptide nucleic acid including the sequence
TABLE-US-00008 (SEQ ID NO: 160)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCAAAGGAGT- lys-lys-lys; or (SEQ ID
NO: 57) lys-lys-lys-TTJJTJT-OOO-TCTCCTTAAACCTGT-lys-lys- lys; or
(SEQ ID NO: 213) lys-lys-lys-TTJJTJT-OOO-TCTCCTTAAACCTGTCTT-lys-
lys-lys (SEQ ID NO: 58)
lys-lys-lys-TJTJTTJT-OOO-TCTTCTCTGTCTCCAC-lys-lys- lys (tc8 16); or
(SEQ ID NO: 59) lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys-
lys-lys; or (SEQ ID NO: 59)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys (SCD-tcPNA
1A); or (SEQ ID NO: 59)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys (SCD-tcPNA
1B); or (SEQ ID NO: 59)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-lys- lys-lys (SCD-tcPNA
1C); or (SEQ ID NO: 209)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC-
lys-lys-lys-NH.sub.2(SCD-tcPNA 1D); or (SEQ ID NO: 209)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC- lys-lys-lys
(SCD-tcPNA 1E); or (SEQ ID NO: 209)
lys-lys-lys-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC- lys-lys-lys
(SCD-tcPNA 1F); or (SEQ ID NO: 60)
lys-lys-lys-TJTJTTJT-OOO-TCTTCTCTGTCTCCACAT-lys- lys-lys;
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA. In even more specific embodiments, the bolded and
underlined residues are miniPEG-containing .gamma.PNA.
c. Exemplary Donors
In some embodiments, the triplex forming molecules are used in
combination with a donor oligonucleotide for correction of IVS2-654
mutation that includes the sequence 5'
AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATCTCTGCATATAAATAT 3'
(SEQ ID NO:65) with the correcting IVS2-654 nucleotide underlined,
or a functional fragment thereof that is suitable and sufficient to
correct the IVS2-654 mutation.
Other exemplary donor sequences include, but are not limited to,
DonorGFP-IVS2-1 (Sense)
5'-GTTCAGCGTGTCCGGCGAGGGCGAGGTGAGTCTATGGGACCCTTGATGTTT-3' (SEQ ID
NO:61), DonorGFP-IVS2-1 (Antisense)
5'-AAACATCAAGGGTCCCATAGACTCACCTCGCCCTCGCCGGACACGCTGAAC-3' (SEQ ID
NO:62), and, or a functional fragment thereof that is suitable and
sufficient to correct a mutation.
In some embodiments, a Sickle Cells Disease mutation can be
corrected using a donor having the sequence
TABLE-US-00009 (SEQ ID NO: 63) ##STR00007##
or a functional fragment thereof that is suitable and sufficient to
correct a mutation, wherein the three boxed nucleotides represent
the corrected codon 6 which reverts the mutant Valine (associated
with human sickle cell disease) back to the wildtype Glutamic acid
and nucleotides in bold font (without underlining) represent
changes to the genomic DNA but not to the encoded amino acid;
or
5ACAGACACCATGGTGCACCTGACTCCTGAGGAGAAGTCTGC CGTTACTGCC 3' (SEQ ID
NO:64), or a functional fragment thereof that is suitable and
sufficient to correct a mutation, wherein the bolded and underlined
residue is the correction (see, e.g., FIG. 6), or
5'T(s)T(s)G(s)CCCCACAGGGCAGTAACGGCAGACTTCTCCTCAGGAGTCAGGTGCACC
ATGGTGTCTGTT(s)T(s)G(s)3' (SEQ ID NO:204), or a functional fragment
thereof that is suitable and sufficient to correct a mutation,
wherein the bolded and underlined residue is the correction and
"(s)" indicates an optional phosphorothioate internucleoside
linkage.
2. Cystic Fibrosis
The disclosed compositions and methods can be used to treat cystic
fibrosis. Cystic fibrosis (CF) is a lethal autosomal recessive
disease caused by defects in the cystic fibrosis transmembrane
conductance regulator (CFTR), an ion channel that mediates Cl-
transport. Lack of CFTR function results in chronic obstructive
lung disease and premature death due to respiratory failure,
intestinal obstruction syndromes, exocrine and endocrine pancreatic
dysfunction, and infertility (Davis, et al., Pediatr Rev.,
22(8):257-64 (2001)). The most common mutation in CF is a three
base-pair deletion (F508del) resulting in the loss of a
phenylalanine residue, causing intracellular degradation of the
CFTR protein and lack of cell surface expression (Davis, et al., Am
J Respir Crit Care Med., 173(5):475-82 (2006)). In addition to this
common mutation there are many other mutations that occur and lead
to disease including a class of mutations due to premature stop
codons, nonsense mutations. In fact nonsense mutations account for
approximately 10% of disease causing mutations. Of the nonsense
mutations G542X and W1282X are the most common with frequencies of
2.6% and 1.6% respectfully.
Although CF is one of the most rigorously characterized genetic
diseases, current treatment of patients with CF focuses on
symptomatic management rather than primary correction of the
genetic defect. Gene therapy has remained an elusive target in CF,
because of challenges of in vivo delivery to the lung and other
organ systems (Armstrong, et al., Archives of disease in childhood
(2014) doi: 10.1136/archdischild-2012-302158. PubMed PMID:
24464978). In recent years, there have been many advances in gene
therapy for treatment of diseases involving the hematolymphoid
system, where harvest and ex vivo manipulation of cells for
autologous transplantation is possible: some examples include the
use of zinc finger nucleases targeting CCR5 to produce HIV-1
resistant cells (Holt, et al., Nature biotechnology, 28(8):839-47
(2010)) correction of the ABCD1 gene by lentiviral vectors for
treatment of adrenoleukodystrophy (Cartier, et al., Science,
326(5954):818-23 (2009)) and correction of SCID due to ADA
deficiency using retroviral gene transfer (Aiuti, et al., The New
England Journal Of Medicine, 360(5):447-58 (2009).
Unfortunately, harvest and autologous transplant is not an option
in CF, due to the involvement of the lung and other internal
organs. As one approach, the UK Cystic Fibrosis Gene Therapy
Consortium has tested liposomes to deliver plasmids containing cDNA
encoding CFTR to the lung (Alton, et al., Thorax, 68(11):1075-7
(2013)), Alton, et al., The Lancet Respiratory Medicine, (2015).
doi: 10.1016/S2213-2600(15)00245-3. PubMed PMID: 26149841.) other
clinical trials have used viral vectors for delivery of the CFTR
gene or CFTR expression plasmids that are compacted by polyethylene
glycol-substituted lysine 30-mer peptides with limited success
(Konstan, et al., Human Gene Therapy, 15(12):1255-69 (2004)).
Moreover, delivery of plasmid DNA for gene addition without
targeted insertion does not result in correction of the endogenous
gene and is not subject to normal CFTR gene regulation, and
virus-mediated integration of the CFTR cDNA could introduce the
risk of non-specific integration into important genomic sites.
However, it has been discovered that triplex-forming PNA molecules
and donor DNA can be used to correct mutations leading to cystic
fibrosis. In preferred embodiments, the compositions are
administered by intranasal or pulmonary delivery. The compositions
can be administered in an effective amount to induce or enhance
gene correction in an amount effective to reduce one or more
symptoms of cystic fibrosis. For example, in some embodiments, the
gene correction occurs at an amount effective to improve impaired
response to cyclic AMP stimulation, improve hyperpolarization in
response to forskolin, reduction in the large lumen negative nasal
potential, reduction in inflammatory cells in the bronchoalveolar
lavage (BAL), improve lung histology, or a combination thereof. In
some embodiments, the target cells are cells, particularly
epithelial cells, that make up the sweat glands in the skin, that
line passageways inside the lungs, liver, pancreas, or digestive or
reproductive systems. In particular embodiments, the target cells
are bronchial epithelial cells. While permanent genomic change
using PNA/DNA is less transient than plasmid-based approaches and
the changes will be passed on to daughter cells, some modified
cells may be lost over time with regular turnover of the
respiratory epithelium. In some embodiments, the target cells are
lung epithelial progenitor cells. Modification of lung epithelial
progenitors can induce more long-term correction of phenotype.
Sequences for the human cystic fibrosis transmembrane conductance
regulator (CFTR) are known in the art, see, for example, GenBank
Accession number: AH006034.1, and compositions and methods of
targeted correction of CFTR are described in McNeer, et al., Nature
Communications, 6:6952, (DOI 10.1038/ncomms7952), 11 pages.
a. Exemplary F508del Target Sites
In some embodiments, the triplex-forming molecules are designed to
target the CFTR gene at nucleotides 9,152-9,159 (TTTCCTCT (SEQ ID
NO:70)) or 9,159-9,168 (TTTCCTCTATGGGTAAG (SEQ ID NO:71) of
accession number AH006034.1, or the non-coding strand (e.g., 3'-5'
complementary sequence) corresponding to nucleotides 9,152-9,159 or
9,152-9,168 (e.g., 5'-AGAGGAAA-3' (SEQ ID NO:72), or
5'-CTTACCCATAGAGGAAA-3' (SEQ ID NO:73)).
In some embodiments, the triplex-forming molecules are designed to
target the CFTR gene at nucleotides 9,039-9,046 (5'-AGAAGAGG-3'
(SEQ ID NO:74), or 9,030-9,046 (5'-ATGCCAACTAGAAGAGG-3' (SEQ ID
NO:75)) of accession number AH006034.1, or the non-coding strand
(e.g., 3'-5' complementary sequence) corresponding to nucleotides
(5' CCTCTTCT 3' (SEQ ID NO:76)) or (5' CCTCTTCTAGTTGGCAT 3' (SEQ ID
NO:77).
In some embodiments, the triplex-forming molecules are designed to
target the CFTR gene at nucleotides 8,665-8,683 (CTTTCCCTT (SEQ ID
NO:78)) or 8,665-8,682 (CTTTCCCTTGTATCTTTT (SEQ ID NO:79) of
accession number AH006034.1, or the non-coding strand (e.g., 3'-5'
complementary sequence) corresponding to nucleotides 8,665-8,683 or
8,665-8,682 (e.g., 5'-AAGGGAAAG-3' (SEQ ID NO:80), or 5'-AAAAGATAC
AAGGGAAAG-3' (SEQ ID NO:81)).
In some embodiments, the triplex-forming molecules are designed to
target the W1282X mutation in CFTR gene at the sequence GAAGGAGAAA
(SEQ ID NO:163), AAAAGGAA (SEQ ID NO:164), or AGAAAAAAGG (SEQ ID
NO:165), or the inverse complement thereof. See FIG. 8C.
In some embodiments, the triplex-forming molecules are designed to
target the G542X mutation in CFTR gene at the sequence AGAAAAA (SEQ
ID NO:166), AGAGAAAGA (SEQ ID NO:167), or AAAGAAA (SEQ ID NO:168),
or the inverse complement thereof. See FIG. 9C.
b. Exemplary Triplex Forming Sequences and Donors
i. F508del
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence includes TCTCCTTT (SEQ ID NO:82), preferably
linked to the sequence TTTCCTCT (SEQ ID NO:83) or more preferably
includes TCTCCTTT (SEQ ID NO:82) linked to the sequence
TTTCCTCTATGGGTAAG (SEQ ID NO:84); or
includes TCTTCTCC (SEQ ID NO:85) preferably linked to the sequence
CCTCTTCT (SEQ ID NO:86), or more preferably includes TCTTCTCC (SEQ
ID NO:85) linked to CCTCTTCTAGTTGGCAT (SEQ ID NO:87); or
includes TTCCCTTTC (SEQ ID NO:88), preferable includes the sequence
TTCCCTTTC (SEQ ID NO:88) linked to the sequence CTTTCCCTT (SEQ ID
NO:89), or more preferable includes the sequence TTCCCTTTC (SEQ ID
NO:89) linked to the sequence CTTTCCCTTGTATCTTTT (SEQ ID
NO:90).
In some preferred embodiments, the triplex forming nucleic acid is
a peptide nucleic acid including the sequence TJTJJTTT (SEQ ID
NO:91), linked to the sequence TTTCCTCT (SEQ ID NO:83) or
TTTCCTCTATGGGTAAG (SEQ ID NO:84); or
TJTTJTJJ (SEQ ID NO:216) linked to the sequence CCTCTTCT (SEQ ID
NO:86), or CCTCTTCTAGTTGGCAT (SEQ ID NO:87);
or TTJJJTTTJ (SEQ ID NO:92) linked to the sequence CTTTCCCTT (SEQ
ID NO:89), or CTTTCCCTTGTATCTTTT (SEQ ID NO:90);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments the triplex forming nucleic acid is a
peptide nucleic acid including the sequence is
lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ ID
NO:93) (hCFPNA2); or
lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys-lys-lys (SEQ ID
NO:93); or
lys-lys-lys-TJTTJTJJ-OOO-CCTCTTCTAGTTGGCAT-lys-lys-lys (SEQ ID
NO:94) (hCFPNA1); or
lys-lys-lys-TTJJJTTTJ-OOO-CTTTCCCTTGTATCTTTT-lys-lys-lys (SEQ ID
NO:95) (hCFPNA3);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA. In even more specific embodiments, the bolded and
underlined residues are miniPEG-containing .gamma.PNA.
In some embodiments, a donor that can be used for CFTR gene
correction, particularly in combination with the foregoing triplex
forming molecules, includes the sequence
5'TTCTGTATCTATATTCATCATAGGAAACACCAAAGATAATGTTCTCC TTAATGGTGCCAGG3'
(SEQ ID NO:96), or a functional fragment thereof that is suitable
and sufficient to correct the F508del mutation in the cystic
fibrosis transmembrane conductance regulator (CFTR) gene.
ii. W1282 Mutation Site
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence CTTCCTCTTT (SEQ ID NO:97), preferable
includes the sequence CTTCCTCTTT (SEQ ID NO:97) linked to the
sequence TTTCTCCTTC (SEQ ID NO:98), or more preferable includes the
sequence CTTCCTCTTT (SEQ ID NO:97) linked to the sequence
TTTCTCCTTCAGTGTTCA (SEQ ID NO:99); or
the triplex-forming molecule includes the nucleic acid sequence
TTTTCCT (SEQ ID NO:100), preferable includes the sequence TTTTCCT
(SEQ ID NO:100) linked to the sequence TCCTTTT (SEQ ID NO:101), or
more preferable includes the sequence TTTTCCT (SEQ ID NO:100)
linked to the sequence TCCTTTTGCTCACCTGTGGT (SEQ ID NO:102); or
the triplex-forming molecule includes the nucleic acid sequence
TCTTTTTTCC (SEQ ID NO:103), preferable includes the sequence
TCTTTTTTCC (SEQ ID NO:103) linked to the sequence CCTTTTTTCT (SEQ
ID NO:104), or more preferable includes the sequence TCTTTTTTCC
(SEQ ID NO:103) linked to the sequence CCTTTTTTCTGGCTAAGT (SEQ ID
NO:105).
In preferred embodiments, the triple forming nucleic acid is a
peptide nucleic acid including the sequence JTTJJTJTTT (SEQ ID
NO:106) linked to the sequence TTTCTCCTTC (SEQ ID NO:98) or
TTTCTCCTTCAGTGTTCA (SEQ ID NO:99); or
a peptide nucleic acid including the sequence TTTTJJT (SEQ ID
NO:107) linked to the sequence TCCTTTT (SEQ ID NO:101) or linked to
the sequence TCCTTTTGCTCACCTGTGGT (SEQ ID NO:102); or
a peptide nucleic acid including the sequence TJTTTTTTJJ (SEQ ID
NO:108) linked to the sequence CCTTTTTTCT (SEQ ID NO:104) or linked
to the sequence CCTTTTTTCTGGCTAAGT (SEQ ID NO:105);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments, the triplex forming nucleic acid is a
peptide nucleic acid including the sequence
lys-lys-lys-JTTJJTJTTT-OOO-TTTCTCCTTCAGTGTTCA-lys-lys-lys (SEQ ID
NO:155) (tcPNA-1236); or
lys-lys-lys-TTTTJJT-OOO-TCCTTTTGCTCACCTGTGGT-lys-lys-lys (SEQ ID
NO:156) (tcPNA-1314); or
lys-lys-lys-TJTTTTTTJJ-OOO-CCTTTTTTCTGGCTAAGT-lys-lys-lys (SEQ ID
NO:157) (tcPNA-1329);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA. In even more specific embodiments, the bolded and
underlined residues are miniPEG-containing .gamma.PNA.
In some embodiments, a donor that can be used for CFTR gene
correction, particularly in combination with the foregoing triplex
forming molecules, includes the sequence
T(s)C(s)T(s)-TGGGATTCAATAACCTTGCAGACAGTGGAG {square root over
(G)}AAGGCCTTTGGCGTGATACCACAGG-(s)T(s)G(s) (SEQ ID NO:109) or a
functional fragment thereof that is suitable and sufficient to
correct a mutation in CFTR, wherein the bolded and underlined
nucleotides are inserted mutations for gene correction, and "(s)"
indicates an optional phosphorothioate internucleoside linkage. See
also, FIGS. 8A-8C, W1282X.
iii. G542X Mutation Site
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence TCTTTTT (SEQ ID NO:110), preferable includes
the sequence TCTTTTT (SEQ ID NO:110) linked to the sequence TTTTTCT
(SEQ ID NO:111), or more preferable includes the sequence TCTTTTT
(SEQ ID NO:110) linked to the sequence TTTTTCTGTAATTTTTAA (SEQ ID
NO:112); or
the triplex-forming molecule includes the nucleic acid sequence
TCTCTTTCT (SEQ ID NO:113), preferable includes the sequence
TCTCTTTCT (SEQ ID NO:113) linked to the sequence TCTTTCTCT (SEQ ID
NO:114), or more preferable includes the sequence TCTCTTTCT (SEQ ID
NO:113) linked to the sequence TCTTTCTCTGCAAACTT (SEQ ID NO:115);
or
the triplex-forming molecule includes the nucleic acid sequence
TTTCTTT (SEQ ID NO:116), preferable includes the sequence TTTCTTT
(SEQ ID NO:116) linked to the sequence TTTCTTT (SEQ ID NO:116), or
more preferable includes the sequence TTTCTTT (SEQ ID NO:116)
linked to the sequence TTTCTTTAAGAACGAGCA (SEQ ID NO:117).
In preferred embodiments, the triple forming nucleic acid is a
peptide nucleic acid including the sequence TJTTTTT (SEQ ID NO:118)
linked to the sequence TTTTTCT (SEQ ID NO:111) or
TTTTTCTGTAATTTTTAA (SEQ ID NO:112); or
a peptide nucleic acid including the sequence TJTJTTTJT (SEQ ID
NO:119) linked to the sequence TCTTTCTCT (SEQ ID NO:114) or linked
to the sequence TCTTTCTCTGCAAACTT (SEQ ID NO:115); or
a peptide nucleic acid including the sequence TTTJTTT (SEQ ID
NO:120) linked to the sequence TTTCTTT (SEQ ID NO:116) or linked to
the sequence TTTCTTTAAGAACGAGCA (SEQ ID NO:117);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments, the triplex forming nucleic acid is a
peptide nucleic acid including the sequence
lys-lys-lys-TJTTTTT-OOO-TTTTTCTGTAATTTTTAA-lys-lys-lys (SEQ ID
NO:121) (tcPNA-302); or
lys-lys-lys-TJTJTTTJT-OOO-TCTTTCTCTGCAAACTT-lys-lys-lys (SEQ ID
NO:122) (tcPNA-529); or
lys-lys-lys-TTTJTTT-OOO-TTTCTTTAAGAACGAGCA-lys-lys-lys (SEQ ID
NO:123) (tcPNA-586);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA. In even more specific embodiments, the bolded and
underlined residues are miniPEG-containing .gamma.PNA.
In some embodiments, a donor that can be used for CFTR gene
correction, particularly in combination with the foregoing triplex
forming molecules, includes the sequence
T(s)C(s)C(s)-AAGTTTGCAGAGAAAGATAATATAGTCCTTGGAGAAGGAGGAATCACCCTGAGTGG
A-G(s)G(s)T(s) (SEQ ID NO:124), or a functional fragment thereof
that is suitable and sufficient to correct a mutation in CFTR,
wherein the bolded and underlined nucleotides are inserted
mutations for gene correction, and "(s)" indicates an optional
phosphorothioate internucleoside linkage. See also, FIGS. 9A-9C,
G542X.
3. HIV
The gene editing compositions can be used to treat infections, for
example those caused by HIV.
a. Exemplary Target Sites
The target sequence for the triplex-forming molecules is within or
adjacent to a human gene that encodes a cell surface receptor for
human immunodeficiency virus (HIV). Preferably, the target sequence
of the triplex-forming molecules is within or is adjacent to a
portion of a HIV receptor gene important to its function in HIV
entry into cells, such as sequences that are involved in efficient
expression of the receptor, transport of the receptor to the cell
surface, stability of the receptor, viral binding by the receptor,
or endocytosis of the receptor. Target sequences can be within the
coding DNA sequence of the gene or within introns. Target sequences
can also be within DNA sequences that regulate expression of the
target gene, including promoter or enhancer sequences.
The target sequence can be within or adjacent to any gene encoding
a cell surface receptor that facilitates entry of HIV into cells.
The molecular mechanism of HIV entry into cells involves specific
interactions between the viral envelope glycoproteins (env) and two
target cell proteins, CD4 and the chemokine receptors. HIV cell
tropism is determined by the specificity of the env for a
particular chemokine receptor, a 7 transmembrane-spanning, G
protein-coupled receptor (Steinberger, et al., Proc. Natl. Acad.
Sci. USA. 97: 805-10 (2000)). The two major families of chemokine
receptors are the CXC chemokine receptors and the CC chemokine
receptors (CCR) so named for their binding of CXC and CC
chemokines, respectively. While CXC chemokine receptors
traditionally have been associated with acute inflammatory
responses, the CCRs are mostly expressed on cell types found in
connection with chronic inflammation and T-cell-mediated
inflammatory reactions: eosinophils, basophils, monocytes,
macrophages, dendritic cells, and T cells (Nansen, et al. 2002,
Blood 99:4). In one embodiment, the target sequence is within or
adjacent to the human genes encoding chemokine receptors,
including, but not limited to, CXCR4, CCR5, CCR2b, CCR3, and
CCR1.
In a preferred embodiment, the target sequence is within or
adjacent to the human CCR5 gene. The CCR5 chemokine receptor is the
major co-receptor for R5-tropic HIV strains, which are responsible
for most cases of initial, acute HIV infection. Individuals who
possess a homozygous inactivating mutation, referred to as the A32
mutation, in the CCR5 gene are almost completely resistant to
infection by R5-tropic HIV-1 strains. The A32 mutation produces a
32 base pair deletion in the CCR5 coding region.
Another naturally occurring mutation in the CCR5 gene is the m303
mutation, characterized by an open reading frame single T to A base
pair transversion at nucleotide 303 which indicates a cysteine to
stop codon change in the first extracellular loop of the chemokine
receptor protein at amino acid 101 (C101X) (Carrington et al.
1997). Mutagenesis assays have not detected the expression of the
m303 co-receptor on the surface of CCR5 null transfected cells
which were found to be non-susceptible to HIV-1 R5-isolates in
infection assays (Blanpain, et al. (2000).
Compositions and methods for targeted gene therapy using
triplex-forming oligonucleotides and peptide nucleic acids for
treating infectious diseases such as HIV are described in U.S.
Application No. 2008/050920 and WO 2011/133803. Each provides
sequences of triplex foiming molecules, target sequences, and donor
oligonucleotides that can be utilized in the compositions and
methods provided herein.
For example, individuals having the homozygous .DELTA.32
inactivating mutation in the CCR5 gene display no significant
adverse phenotypes, suggesting that this gene is largely
dispensable for normal human health. This makes the CCR5 gene a
particularly attractive target for targeted mutagenesis using the
triplex-forming molecules disclosed herein. The gene for human CCR5
is known in the art and is provided at GENBANK accession number
NM_000579. The coding region of the human CCR5 gene is provided by
nucleotides 358 to 1416 of GENBANK accession number NM_000579.
In some embodiments, the target region is a polypurine site within
or adjacent to a gene encoding a chemokine receptor including
CXCR4, CCR5, CCR2b, CCR3, and CCR1. In a preferred embodiment, the
target region is a polypurine or homopurine site within the coding
region of the human CCR5 gene. Three homopurine sites in the coding
region of the CCR5 gene that are especially useful as target sites
for triplex-forming molecules are from positions 509-518, 679-690
and 900-908 relative to the ATG start codon.
The homopurine site from 679-690 partially encompasses the site of
the nonsense mutation created by the 432 mutation. Triplex-forming
molecules that bind to this target site are particularly
useful.
b. Exemplary Triplex Forming Sequences
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence CTCTTCTTCT (SEQ ID NO:125), preferable
includes the sequence CTCTTCTTCT (SEQ ID NO:125) linked to the
sequence TCTTCTTCTC (SEQ ID NO:126), or more preferable includes
the sequence CTCTTCTTCT (SEQ ID NO:125) linked to the sequence
TCTTCTTCTCATTTC (SEQ ID NO:127).
In some embodiments, the triplex-forming molecule includes the
nucleic acid sequence CTTCT (SEQ ID NO:128), preferable includes
the sequence CTTCT (SEQ ID NO:128) linked to the sequence TCTTC
(SEQ ID NO:129) or TCTTCTTCTC (SEQ ID NO:130), or more preferable
includes the sequence CTTCT (SEQ ID NO:128) linked to the sequence
TCTTCTTCTCATTTC (SEQ ID NO:131).
In preferred embodiments, the triplex forming nucleic acid is a
peptide nucleic acid including the sequence JTJTTJTTJT (SEQ ID
NO:132) linked to the sequence TCTTCTTCTC (SEQ ID NO:126) or
TCTTCTTCTCATTTC (SEQ ID NO:127);
or JTTJT (SEQ ID NO:133) linked to the sequence TCTTC (SEQ ID
NO:129) or TCTTCTTCTC (SEQ ID NO:130) or more preferably
TCTTCTTCTCATTTC (SEQ ID NO:131);
optionally, but preferably wherein one or more of the PNA monomers
is a .gamma.PNA.
In specific embodiments, the triplex forming nucleic acid is a
peptide nucleic acid including the sequence
Lys-Lys-Lys-JTJTTJTTJT-OOO-TCTTCTTCTCATTTC-Lys-Lys-Lys (SEQ ID
NO:134) (PNA-679);
or Lys-Lys-Lys-JTTJT-OOO-TCTTCTTCTCATTTC-Lys-Lys-Lys (SEQ ID
NO:135) (tcPNA-684) optionally, but preferably wherein one or more
of the PNA monomers is a .gamma.PNA. In even more specific
embodiments, the bolded and underlined residues are
miniPEG-containing .gamma.PNA.
c. Exemplary Donor Sequences
In some embodiments, the triplex forming molecules are used in
combination with one or more donor oligonucleotides such as donor
591 having the sequence: 5' AT TCC CGA GTA GCA GAT GAC CAT GAC AGC
TTA GGG CAG GAC CAG CCC CAA GAT GAC TAT C 3' (SEQ ID NO:136), or
donor 597 having the sequence 5' TT TAG GAT TCC CGA GTA GCA GAT GAC
CCC TCA GAG CAG CGG CAG GAC CAG CCC CAA GAT G 3' (SEQ ID NO:137),
which can be used in combination to induce two different non-sense
mutations, one in each allele of the CCR5 gene, in the vicinity of
the A32 deletion (mutation sites are bolded); or a functional
fragment thereof that is suitable and sufficient to introduce a
non-sense mutation in at least one allele of the CCR5 gene.
In another preferred embodiment, donor oligonucleotides are
designed to span the .DELTA.32 deletion site (see, e.g., FIG. 1 of
WO 2011/133803) and induce changes into a wildtype CCR5 allele that
mimic the .DELTA.32 deletion. Donor sequences designed to target
the .DELTA.32 deletion site may be particularly usefully to
facilitate knockout of the single wildtype CCR5 allele in
heterozygous cells.
Preferred donor sequences designed to target the .DELTA.32 deletion
site include, but are not limited to,
TABLE-US-00010 Donor DELTA32JDC: (SEQ ID NO: 138)
5'GATGACTATCTTTAATGTCTGGAAATTCTTCCAGAATTAATTAAG
ACTGTATGGAAAATGAGAGC 3'; Donor DELTAJDC2: (SEQ ID NO: 139)
5'CCCCAAGATGACTATCTTTAATGTCTGGAACGATCATCAGAATTG
ATACTGACTGTATGGAAAATG 3'; and Donor DELTA32RSB: (SEQ ID NO: 140)
5'GATGACTATCTTTAATGTCTGGAAATTCTACTAGAATTGATACTG
ACTGTATGGAAAATGAGAGC 3',
or a functional fragment of SEQ ID NO:138, 139, or 140 that is
suitable and sufficient to introduce mutation CCR5 gene.
4. Lysosomal Storage Diseases
The disclosed compositions and methods compositions can also be
used to treat lysosomal storage diseases. Lysosomal storage
diseases (LSDs) are a group of more than 50 clinically-recognized,
rare inherited metabolic disorders that result from defects in
lysosomal function (Walkley, J Inherit. Metab. Dis., 32(2):181-9
(2009)). Lysosomal storage disorders are caused by dysfunction of
the cell's lysosome orangelle, which is part of the larger
endosomal/lysosomal system. Together with the ubiquitin-proteosomal
and autophagosomal systems, the lysosome is essential to substrate
degradation and recycling, homeostatic control, and signaling
within the cell. Lysosomal dysfunction is usually the result of a
deficiency of a single enzyme necessary for the metabolism of
lipids, glycoproteins (sugar containing proteins) or
mucopolysaccharides (long unbranched polysaccharides consisting of
a repeating disaccharide unit; also known as glycosaminoglycans, or
GAGs) which are fated for breakdown or recycling. Enzyme deficiency
reduces or prevents break down or recycling of the unwanted lipids,
glycoproteins, and GAGs, and results in buildup or "storage" of
these materials within the cell. Most lysosomal diseases show
widespread tissue and organ involvement, with brain, viscera, bone
and connective tissues often being affected. More than two-thirds
of lysosomal diseases affect the brain. Neurons appear particularly
vulnerable to lysosomal dysfunction, exhibiting a range of defects
from specific axonal and dendritic abnormalities to neuron
death.
Individually, LSDs occur with incidences of less than 1:100,000,
however, as a group the incidence is as high as 1 in 1,500 to 7,000
live births (Staretz-Chacham, et al., Pediatrics, 123(4):1191-207
(2009)). LSDs are typically the result of inborn genetic errors.
Most of these disorders are autosomal recessively inherited,
however a few are X-linked recessively inherited, such as Fabry
disease and Hunter syndrome (MPS II). Affected individuals
generally appear normal at birth, however the diseases are
progressive. Develop of clinical disease may not occur until years
or decades later, but is typically fatal. Lysosomal storage
diseases affect mostly children and they often die at a young and
unpredictable age, many within a few months or years of birth. Many
other children die of this disease following years of suffering
from various symptoms of their particular disorder. Clinical
disease may be manifest as mental retardation and/or dementia,
sensory loss including blindness or deafness, motor system
dysfunction, seizures, sleep and behavioral disturbances, and so
forth. Some people with Lysosomal storage disease have enlarged
livers (hepatomegaly) and enlarged spleens (splenomegaly),
pulmonary and cardiac problems, and bones that grow abnormally.
Treatment for many LSDs is enzyme replacement therapy (ERT) and/or
substrate reduction therapy (SRT), as wells as treatment or
management of symptoms. The average annual cost of ERT in the
United States ranges from $90,000 to $565,000. While ERT has
significant systemic clinical efficacy for a variety of LSDs,
little or no effects are seen on central nervous system (CNS)
disease symptoms, because the recombinant proteins cannot penetrate
the blood-brain barrier. Allogeneic hematopoietic stem cell
transplantation (HSCT) represents a highly effective treatment for
selected LSDs. It is currently the only means to prevent the
progression of associated neurologic sequelae. However, HSCT is
expensive, requires an HLA-matched donor and is associated with
significant morbidity and mortality. Recent gene therapy studies
suggest that LSDs are good targets for this type of treatment.
Compositions and methods for targeted gene therapy using
triplex-forming oligonucleotides and peptide nucleic acids for
treating lysosomal storage diseases are described in WO
2011/133802, which provides sequences of triplex forming molecules,
target sequences, and donor oligonucleotides that can be utilized
in the compositions and methods provided herein.
For example, the disclosed compositions and methods can be are
employed to treat Gaucher's disease (GD). Gaucher's disease, also
known as Gaucher syndrome, is the most common lysosomal storage
disease. Gaucher's disease is an inherited genetic disease in which
lipid accumulates in cells and certain organs due to deficiency of
the enzyme glucocerebrosidase (also known as acid
.beta.-glucosidase) in lysosomes. Glucocerebrosidase enzyme
contributes to the degradation of the fatty substance
glucocerebroside (also known as glucosylceramide) by cleaving
b-glycoside into b-glucose and ceramide subunits (Scriver C R,
Beaudet A L, Valle D, Sly W S. The metabolic and molecular basis of
inherited disease. 8th ed. New York: McGraw-Hill Pub, 2001:
3635-3668). When the enzyme is defective, the substance
accumulates, particularly in cells of the mononuclear cell lineage,
and organs and tissues including the spleen, liver, kidneys, lungs,
brain and bone marrow.
There are two major forms: non-neuropathic (type 1, most commonly
observed type in adulthood) and neuropathic (type 2 and 3). GBA
(GBA glucosidase, beta, acid), the only known human gene
responsible for glucosidase-mediated GD, is located on chromosome
1, location 1q21. More than 200 mutations have been defined within
the known genomic sequence of this single gene (NCBI Reference
Sequence: NG_009783.1). The most commonly observed mutations are
N370S, L444P, RecNcil, 84GG, R463C, recTL and 84 GG is a null
mutation in which there is no capacity to synthesize enzyme.
However, N370S mutation is almost always related with type 1
disease and milder forms of disease. Very rarely, deficiency of
sphingolipid activator protein (Gaucher factor, SAP-2, saposin C)
may result in GD. In some embodiments, triplex-forming molecules
are used to induce recombination of donor oligonucleotides designed
to correct mutations in GBA.
In another embodiment, compositions and the methods disclosed
herein are used to treat Fabry disease (also known as Fabry's
disease, Anderson-Fabry disease, angiokeratoma corporis diffusum
and alpha-galactosidase A deficiency), a rare X-linked recessive
disordered, resulting from a deficiency of the enzyme alpha
galactosidase A (a-GAL A, encoded by GLA). The human gene encoding
GLA has a known genomic sequence (NCBI Reference Sequence:
NG_007119.1) and is located at Xp22 of the X chromosome. Mutations
in GLA result in accumulation of the glycolipid
globotriaosylceramide (abbreviated as Gb3, GL-3, or ceramide
trihexoside) within the blood vessels, other tissues, and organs,
resulting in impairment of their proper function (Karen, et al.,
Dermatol. Online J., 11 (4): 8 (2005)). The condition affects
hemizygous males (i.e. all males), as well as homozygous, and
potentially heterozygous (carrier), females. Males typically
experience severe symptoms, while women can range from being
asymptomatic to having severe symptoms. This variability is thought
to be due to X-inactivation patterns during embryonic development
of the female. In some embodiments, triplex-forming molecules are
used to induce recombination of donor oligonucleotides designed to
correct mutations in GLA.
In preferred embodiments, the disclosed compositions and methods
are used to treat Hurler syndrome (HS). Hurler syndrome, also known
as mucopolysaccharidosis type I (MPS I), .alpha.-L-iduronidase
deficiency, and Hurler's disease, is a genetic disorder that
results in the buildup of mucopolysaccharides due to a deficiency
of .alpha.-L iduronidase, an enzyme responsible for the degradation
of mucopolysaccharides in lysosomes (Dib and Pastories, Genet. Mol.
Res., 6(3):667-74 (2007)). MPS I is divided into three subtypes
based on severity of symptoms. All three types result from an
absence of, or insufficient levels of, the enzyme
.alpha.-L-iduronidase. MPS I H or Hurler syndrome is the most
severe of the MPS I subtypes. The other two types are MPS I S or
Scheie syndrome and MPS I H-S or Hurler-Scheie syndrome. Without
.alpha.-L-iduronidase, heparan sulfate and dermatan sulfate, the
main components of connective tissues, build-up in the body.
Excessive amounts of glycosaminoglycans (GAGs) pass into the blood
circulation and are stored throughout the body, with some excreted
in the urine. Symptoms appear during childhood, and can include
developmental delay as early as the first year of age. Patients
usually reach a plateau in their development between the ages of
two and four years, followed by progressive mental decline and loss
of physical skills (Scott et al., Hum. Mutat. 6: 288-302 (1995)).
Language may be limited due to hearing loss and an enlarged tongue,
and eventually site impairment can results from clouding of cornea
and retinal degeneration. Carpal tunnel syndrome (or similar
compression of nerves elsewhere in the body) and restricted joint
movement are also common.
a. Exemplary Target Sites
The human gene encoding alpha-L-iduronidase (.alpha.-L-iduronidase;
IDUA) is found on chromosome 4, location 4p16.3, and has a known
genomic sequence (NCBI Reference Sequence: NG 008103.1). Two of the
most common mutations in IDUA contributing to Hurler syndrome are
the Q70X and the W420X, non-sense point mutations found in exon 2
(nucleotide 774 of genomic DNA relative to first nucleotide of
start codon) and exon 9 (nucleotide 15663 of genomic DNA relative
to first nucleotide of start codon) of IDUA respectively. These
mutations cause dysfunction alpha-L-iduronidase enzyme. Two
triplex-forming molecule target sequences including a
polypurine:polypyrimidine stretches have been identified within the
IDUA gene. One target site with the polypurine sequence 5'
CTGCTCGGAAGA 3' (SEQ ID NO:141) and the complementary
polypyrimidine sequence 5' TCTTCCGAGCAG 3' (SEQ ID NO:142) is
located 170 base pairs downstream of the Q70X mutation. A second
target site with the polypurine sequence 5' CCTTCACCAAGGGGA 3' (SEQ
ID NO:143) and the complementary polypyrimidine sequence 5'
TCCCCTTGGTGAAGG 3' (SEQ ID NO:144) is located 100 base pairs
upstream of the W402X mutation. In preferred embodiments,
triplex-forming molecules are designed to bind/hybridize in or near
these target locations.
b. Exemplary Triplex Forming Sequences and Donors
i. W402X mutation
In some embodiments, a triplex-forming molecule binds to the target
sequence upstream of the W402X mutation includes the nucleic acid
sequence TTCCCCT (SEQ ID NO:145), preferable includes the sequence
TTCCCCT (SEQ ID NO:145) linked to the sequence TCCCCTT (SEQ ID
NO:146), or more preferable includes the sequence TTCCCCT (SEQ ID
NO:145) linked to the sequence TCCCCTTGGTGAAGG (SEQ ID NO:147).
In some preferred embodiments, the triplex forming nucleic acid is
a peptide nucleic acid that binds to the target sequence upstream
of the W402X mutation including the sequence TTJJJJT (SEQ ID
NO:148), linked to the sequence TCCCCTT (SEQ ID NO:146) or
TCCCCTTGGTGAAGG (SEQ ID NO:147), optionally, but preferably wherein
one or more of the PNA monomers is a .gamma.PNA.
In specific embodiments, the triplex forming nucleic acid is a
peptide nucleic acid having the sequence
Lys-Lys-Lys-TTJJJJT-OOO-TCCCCTTGGTGAAGG-Lys-Lys-Lys (SEQ ID NO:159)
(IDUA402tc715) optionally, but preferably wherein one or more of
the PNA monomers is a .gamma.PNA. In even more specific
embodiments, the bolded and underlined residues are
miniPEG-containing .gamma.PNA.
In the most preferred embodiments, triplex-forming molecules are
administered according to the disclosed methods in combination with
one or more donor oligonucleotides designed to correct the point
mutations at Q70X or W402X mutations sites. In some embodiments, in
addition to containing sequence designed to correct the point
mutation at Q70X or W402X mutation, the donor oligonuclotides may
also contain 7 to 10 additional, synonymous (silent) mutations. The
additional silent mutations can facilitate detection of the
corrected target sequence using allele-specific PCR of genomic DNA
isolated from treated cells.
In some embodiments, the donor oligonucleotide with the sequence 5'
AGGACGGTCCCGGCCTGCGACACTTCCGCCCATAATTGTTCTTCATCT GCGGGGCGGGGGGGGG
3' (SEQ ID NO:149), or a functional fragment thereof that is
suitable and sufficient to correct the W402X mutation is
administered with triplex-forming molecules designed to target the
binding site upstream of W402X to correct the W402X mutation in
cells.
ii. Q70X Mutation
In some embodiments, a triplex-forming molecule that binds to the
target sequence downstream of the Q70X mutation includes the
nucleic acid sequence CCTTCT (SEQ ID NO:150), preferable includes
the sequence CCTTCT (SEQ ID NO:150) linked to the sequence TCTTCC
(SEQ ID NO:151), or more preferable includes the sequence CCTTCT
(SEQ ID NO:150) linked to the sequence TCTTCCGAGCAG (SEQ ID
NO:152).
In preferred embodiments, the triplex forming nucleic acid is a
peptide nucleic acid that binds to the target sequence downstream
of the Q70X mutation including the sequence JJTTJT (SEQ ID NO:153)
linked to the sequence TCTTCC (SEQ ID NO:151) or TCTTCCGAGCAG (SEQ
ID NO:152) optionally, but preferably wherein one or more of the
PNA monomers is a .gamma.PNA.
In a specific embodiment, a tcPNA with a sequence of
Lys-Lys-Lys-JJTTJT-OOO-TCTTCCGAGCAG-Lys-Lys-Lys (SEQ ID NO:153)
(IDUA402tc715) optionally, but preferably wherein one or more of
the PNA monomers is a .gamma.PNA. In even more specific
embodiments, the bolded and underlined residues are
miniPEG-containing .gamma.PNA.
A donor oligonucleotide can have the sequence
5'GGGACGGCGCCCACATAGGCCAAATTCAATTGCTGATCCCAGCTTA
AGACGTACTGGTCAGCCTGGC 3' (SEQ ID NO:154), or a functional fragment
thereof that is suitable and sufficient to correct the Q70X
mutation is administered with triplex-forming molecules designed to
target the binding site downstream of Q70X to correct the of Q70X
mutation in cells.
X. Combination Therapies
Each of the different components of gene editing and potentiation
disclosed here can be administered alone or in any combination and
further in combination with one or more additional active agents.
In all cases, the combination of agents can be part of the same
admixture, or administered as separate compositions. In some
embodiments, the separate compositions are administered through the
same route of administration. In other embodiments, the separate
compositions are administered through different routes of
administration.
A. Conventional Therapeutic Agents
Examples of preferred additional active agents include other
conventional therapies known in the art for treating the desired
disease or condition. For example, in the treatment of sickle cell
disease, the additional therapy may be hydroxurea.
In the treatment of cystic fibrosis, the additional therapy may
include mucolytics, antibiotics, nutritional agents, etc. Specific
drugs are outlined in the Cystic Fibrosis Foundation drug pipeline
and include, but are not limited to, CFTR modulators such as
KALYDECO.RTM. (invascaftor), ORKAMBI.TM. (lumacaftor+ivacaftor),
ataluren (PTC124), VX-661+ invacaftor, riociguat, QBW251, N91115,
and QR-010; agents that improve airway surface liquid such as
hypertonic saline, bronchitol, and P-1037; mucus alteration agents
such as PULMOZYME.RTM. (dornase alfa); anti-inflammatories such as
ibuprofen, alpha 1 anti-trypsin, CTX-4430, and JBT-101;
anti-infective such as inhaled tobramycin, azithromycin,
CAYSTON.RTM. (aztreonam for inhalation solution), TOBI inhaled
powder, levofloxacin, ARIKACE.RTM. (nebulized liposomal amikacin),
AEROVANC.RTM. (vancomycin hydrochloride inhalation powder), and
gallium; and nutritional supplements such as aquADEKs, pancrelipase
enzyme products, liprotamase, and burlulipase.
In the treatment of HIV, the additional therapy maybe an
antiretroviral agents including, but not limited to, a
non-nucleoside reverse transcriptase inhibitor (NNRTIs), a
nucleoside reverse transcriptase inhibitor (NRTIs), a protease
inhibitors (PIs), a fusion inhibitors, a CCR5 antagonists (CCR5s)
(also called entry inhibitors), an integrase strand transfer
inhibitors (INSTIs), or a combination thereof.
In the treatment of lysosomal storage disease, the additional
therapy could include, for example, enzyme replacement therapy,
bone marrow transplantation, or a combination thereof.
B. Additional Mutagenic Agents
The compositions can be used in combination with other mutagenic
agents. In a preferred embodiment, the additional mutagenic agents
are conjugated or linked to gene editing technology or a delivery
vehicle (such as a nanoparticle) thereof. Additional mutagenic
agents that can be used in combination with gene editing
technology, particularly triplex forming molecules, include agents
that are capable of directing mutagenesis, nucleic acid
crosslinkers, radioactive agents, or alkylating groups, or
molecules that can recruit DNA-damaging cellular enzymes. Other
suitable mutagenic agents include, but are not limited to, chemical
mutagenic agents such as alkylating, bialkylating or intercalating
agents. A preferred agent for co-administration is psoralen-linked
molecules as described in PCT/US/94/07234 by Yale University.
It may also be desirable to administer gene editing compositions in
combination with agents that further enhance the frequency of gene
modification in cells. For example, the disclosed compositions can
be administered in combination with a histone deacetylase (HDAC)
inhibitor, such as suberoylanilide hydroxamic acid (SAHA), which
has been found to promote increased levels of gene targeting in
asynchronous cells.
The nucleotide excision repair pathway is also known to facilitate
triplex-forming molecule-mediated recombination. Therefore, the
disclosed compositions can be administered in combination with an
agent that enhances or increases the nucleotide excision repair
pathway, for example an agent that increases the expression, or
activity, or localization to the target site, of the endogenous
damage recognition factor XPA.
Compositions may also be administered in combination with a second
active agent that enhances uptake or delivery of the gene editing
technology. For example, the lysosomotropic agent chloroquine has
been shown to enhance delivery of PNAs into cells (Abes, et al., J
Controll. Rel., 110:595-604 (2006). Agents that improve the
frequency of gene modification are particularly useful for in vitro
and ex vivo application, for example ex vivo modification of
hematopoietic stem cells for therapeutic use.
XI. Methods for Determining Triplex Formation and Gene
Modification
A. Methods for Determining Triplex Formation
A useful measure of triple helix formation is the equilibrium
dissociation constant, K.sub.d, of the triplex, which can be
estimated as the concentration of triplex-forming molecules at
which triplex formation is half-maximal. Preferably, the molecules
have a binding affinity for the target sequence in the range of
physiologic interactions. Preferred triplex-forming molecules have
a K.sub.d less than or equal to approximately 10.sup.-7 M. Most
preferably, the K.sub.d is less than or equal to 2.times.10.sup.-8
M in order to achieve significant intramolecular interactions. A
variety of methods are available to determine the K.sub.d of
triplex-forming molecules with the target duplex. In the examples
which follow, the K.sub.d was estimated using a gel mobility shift
assay (R. H. Durland et al., Biochemistry 30, 9246 (1991)). The
dissociation constant (K.sub.d) can be determined as the
concentration of triplex-forming molecules in which half was bound
to the target sequence and half was unbound.
B. Methods for Determining Gene Modification
Sequencing and allele-specific PCR are preferred methods for
determining if gene modification has occurred. PCR primers are
designed to distinguish between the original allele, and the new
predicted sequence following recombination. Other methods of
determining if a recombination event has occurred are known in the
art and may be selected based on the type of modification made.
Methods include, but are not limited to, analysis of genomic DNA,
for example by sequencing, allele-specific PCR, or restriction
endonuclease selective PCR (REMS-PCR); analysis of mRNA transcribed
from the target gene for example by Northern blot, in situ
hybridization, real-time or quantitative reverse transcriptase (RT)
PCT; and analysis of the polypeptide encoded by the target gene,
for example, by immunostaining, ELISA, or FACS. In some cases,
modified cells will be compared to parental controls. Other methods
may include testing for changes in the function of the RNA
transcribed by, or the polypeptide encoded by the target gene. For
example, if the target gene encodes an enzyme, an assay designed to
test enzyme function may be used.
XII. Kits
Medical kits are also disclosed. The medical kits can include, for
example, a dosage supply of gene editing technology or a
potentiating agent thereof, or a combination thereof in separately
or together in the same admixture. The active agents can be
supplied alone (e.g., lyophilized), or in a pharmaceutical
composition. The active agents can be in a unit dosage, or in a
stock that should be diluted prior to administration. In some
embodiments, the kit includes a supply of pharmaceutically
acceptable carrier. The kit can also include devices for
administration of the active agents or compositions, for example,
syringes. The kits can include printed instructions for
administering the compound in a use as described above.
EXAMPLES
Example 1: Triplex-Forming PNA Design and Nanoparticle Formulation
for Gene Editing of a .beta.-Globin Mutation
Materials and Methods
Oligonucleotides
.sup.MP.gamma.PNA monomers were prepared as reported (Sahu, et al.,
J. Org. Chem., 76:5614-5627 (2011)). PNA oligomers were synthesized
on solid support using Boc chemistry, as described (Bahal, et al.,
ChemBioChem, 13:56-60 (2012)). The sequences of PNAs used in this
study are:
TABLE-US-00011 tcPNA1: (SEQ ID NO: 33)
H-KKK-JTTTJTTTJTJT-OOO-TCTCTTTCTTTCAGGGCA-KKK- NH.sub.2 tcPNA2:
(SEQ ID NO: 34) H-KKK-TTTTJJJ------OOO-CCCTTTTGCTAATCATGT-KKK-
NH.sub.2 tcPNA3: (SEQ ID NO: 35)
H-KKK-TTTJTJJ------OOO-CCTCTTTGCACCATTCT-KKK- NH.sub.2
.gamma.tcPNA4: (SEQ ID NO: 162)
H-KKK-JTTTJTTTJTJT-OOO-TCTCTTTCTTTCAGGGCA-KKK- NH.sub.2
.gamma.tcPNA4-Scr.: (SEQ ID NO: 158)
H-KKK-TTJTTTJTTJTJ-OOO-CTCTTCTTTCTTGACAGG-KKK- NH.sub.2
Sequences of tcPNAs and .gamma.tcPNAs used in this study to bind to
positions 577 to 595 (tcPNA1 and .gamma.tcPNA4), 611 to 629
(tcPNA2), and 807 to 825 (tcPNA3) in .beta.-globin intron 2 within
the .beta.-globin/GFP fusion gene and within the human
.beta.-globin gene in the thalassemic mouse model.
.gamma.tcPNA4-Scr is a scrambled version of .gamma.tcPNA4 with the
same base composition. Bold and underline indicates .gamma.PNA
residues. All PNAs have three lysine residues conjugated to each
end. "J" indicates pseudoisocytosine substituted for C to allow
pH-independent triplex formation. "O" represents
8-amino-2,6,10-trioxaoctanoic acid residues that are used to form
flexible linkers connecting the Hoogsteen and Watson-Crick binding
domains of the tcPNAs.
The single-stranded donor DNA oligomer was prepared by standard DNA
synthesis except for the inclusion of 3 phosphorothioate
internucleoside linkages at each end to protect from nuclease
degradation. The sequence of the donor DNA matches positions 624 to
684 in .beta.-globin intron 2 and is as follows, with the
correcting IVS2-654 nucleotide underlined:
TABLE-US-00012 (SEQ ID NO: 65)
5'AAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGCAATATC
TCTGCATATAAATAT3'.
PLGA Nanoparticle Synthesis and Characterization
PLGA nanoparticles containing the PNAs and DNAs were formulated
using a double-emulsion solvent evaporation method and
characterized as previously described (McNeer, et al., Molecular
Therapy, 19(1):172-180 (2011), and). Release profiles were analyzed
as previously described (McNeer, et al., Mol. Ther., 19:172-180
(2011)).
DNA Binding Gel Shift Assays
For gel electrophoresis, synthetic 120 bp dsDNA targets were
incubated with indicated oligomers at 37 C in low ionic strength
buffer (10 mM NaPi, pH 7.4). The samples were separated on 10%
non-denaturing polyacrylamide gels in 1.times.TBE buffer. The gels
were run at 100 V/cm for 1.5 hr. After electrophoresis, the gels
were stained with 1.times.SYBR-Gold (catalog #S11494, Invitrogen)
for 10 min, washed 2.times. with 1.times.TBE buffer, and then
imaged using a gel documentation system (BioDoc-It System). The
images were then inverted using Adobe Photoshop 6.0.
Results
To assay for gene editing in a robust and quantitative manner, a
transgenic mouse model was utilized with a .beta.-globin/GFP fusion
transgene of human .beta.-globin intron 2 carrying a
thalassemia-associated IVS2-654 (C.fwdarw.T) mutation embedded
within the GFP coding sequence, resulting in incorrect splicing of
.beta.-globin/GFP mRNA and lack of GFP expression (Sazani, et al.,
Nat. Biotechnol., 20:1228-1233 (2002)). PNA-mediated
triplex-formation induces DNA repair and recombination of the
genomic site with a 60-nucleotide sense donor DNA that is
homologous to a portion of the .beta.-globin intron 2 sequence
except for providing a wild-type nucleotide at the IVS2-654
position. Via recombination, the splice-site mutation is corrected
and expression of functional GFP occurs (FIG. 1A) (McNeer, et al.,
Gene Therapy, 20:658-669 (2013); Bahal, et al., Curr. Gene Ther.,
14:331-342 (2014)). Hence, GFP expression provides a direct
phenotypic assessment of genome editing frequencies that can be
quantified by flow cytometry.
A series of tcPNAs were designed to bind to selected polypurine
stretches in the .beta.-globin intron in the vicinity of the
IVS2-654 mutation (FIG. 1B). Two of the tcPNAs were synthesized to
contain partial substitution with a mini-polyethylene-glycol
(mini-PEG) group at the .gamma. position (.sup.MP.gamma.PNA) (FIG.
1C, and sequences above). Gamma substitutions in PNAs have been
shown to enhance strand invasion and DNA binding affinity in the
Watson-Crick binding mode due to helical pre-organization enforced
by the modification (Bahal, et al., ChemBioChem, 13:56-60 (2012)).
.gamma.tcPNA4 matches the sequence of tcPNA1 except that it
contains .gamma. units at alternating positions in the Watson-Crick
domain (see sequences above). Scrambled .gamma.tcPNA
(.gamma.tcPNA4-Scr) had the same base composition as .gamma.tcPNA4
but a scrambled sequence. All tcPNA oligomers were synthesized with
3 lysines at both termini to improve solubility and increase
binding affinity to genomic DNA (see sequences above).
Gel shift assays to assess the binding of the tcPNAs to 120-bp DNA
duplexes containing the respective target sequences showed that all
of the tcPNAs bound specifically to their target sites in duplex
DNA under physiological conditions. No binding was seen in the case
of the scrambled sequence .gamma.tcPNA4-Scr oligomer.
Poly(lactic-co-glycolic acid) (PLGA) NPs can effectively deliver
PNA/donor DNA combinations into primary human and mouse
hematopoietic cells with essentially no toxicity (McNeer, et al.,
Gene Therapy, 20:658-669 (2013); Schleifinan, et al., Mol.
Ther.--Nucleic Acids, 2:e135 (2013); McNeer, et al., Mol. Ther.,
19:172-180 (2011)). Here, tcPNAs and donor DNAs, at a molar ratio
of 2:1, were incorporated into PLGA NPs. The NP formulations were
evaluated by scanning electron microscopy (SEM) and dynamic light
scattering (DLS). All the NPs exhibited sizes within the expected
range and showed a uniform charge distribution as calculated from
their zeta potential.
TABLE-US-00013 TABLE 1 Hydrodynamic diameter of formulated PLGA
nanoparticles measured using dynamic light scattering in PBS
buffer. Sample Diameter (nm) tcPNA 1/donor DNA 293.1 .+-. 6.1 tcPNA
2/donor DNA 610.6 .+-. 27.7 tcPNA 3/donor DNA 373.0 .+-. 4.3
.gamma.tcPNA 4/donor DNA 291.0 .+-. 4.7 .gamma.tcPNA 4-Scr/donor
DNA 458.6 .+-. 8.2 Donor DNA 907.3 .+-. 200
TABLE-US-00014 TABLE 2 Zeta potential of formulated PLGA
nanoparticles. Sample Zeta Potential (mV) tcPNA1/donor DNA -24.6
.+-. 0.4 tcPNA2/donor DNA -16.5 .+-. 0.5 tcPNA3/donor DNA -23.6
.+-. 0.5 .gamma.tcPNA4/donor DNA -23.4 .+-. 0.5 .gamma.tcPNA
4-Scr/donor DNA -19.5 .+-. 1.3 Donor DNA -29.1 .+-. 0.4
Nucleic acid release profiles in aqueous solution were consistent
with previous studies, indicating no deleterious impact of the
.gamma. modifications on release from NPs (FIG. 1E).
Example 2: .gamma.tcPNA Edit Bone Marrow Cell Genome Ex Vivo
Materials and Methods
Ex Vivo Experiments
Bone marrow cells were harvested by flushing of femurs and tibias
from .beta.-globin/GFP transgenic mice with Roswell Park Memorial
Institute (RPMI)/10% FBS media. Two mg/ml of nanoparticles were
used to treat approximately 300,000-500,000 cells for 48 hr in
RPMI/10% FBS media containing glutamine, in triplicate samples.
After 48 hr, cells were fixed by using 4% paraformaldehyde, and
flow cytometry analyses were performed. Cells treated with blank
nanoparticles were included as a control.
For CD117+ cell experiments, Iscove's Modified Dulbecco's Media
(IMDM) media containing insulin (10 ng/ml), FCS (10%) and
erythropoietin (1 U/ml) was used to culture CD117+ cells after
isolation using magnetic separation. Where indicated, 3 .mu.g/ml of
SCF (Recombinant murine SCF, catalog #250-03, PeproTech, Rocky
Hill, N.J.;) was added prior to nanoparticle treatment. 2 mg/ml of
NPs were used to treat 50,000-100,000 CD117+ cells in triplicate
for 48 hrs in the above media, followed by flow cytometry analyses
as above. Inhibitors were used at concentrations of 200 nM
(dasatinib), 1.0 .mu.M (MEK162) and 3.0 .mu.M (BKM120). Dasatanib
was obtained from Cayman Chemical (Ann Arbor, Mich.; item #11498)
and dissolved according to manufacturer's protocol. MEK162 and
BKM120 were obtained from Dr. Harriet Kluger, Yale University.
Comet Assay
400,000 bone marrow cells/well were plated on 6-well plates in 1 mL
media, then treated with 2 mg/mL of PLGA nanoparticles with or
without PNA and donor DNA. After 48 hours, cells were scraped and
harvested, and prepared using the Trevigen Comet Assay kit per
manufacturer's protocol (Trevigen, Gaithersburg, Md.). Briefly,
cells were suspended in agarose, added to comet slides, allowed to
set, incubated 1 hr in lysis solution, placed in electrophoresis
solution for 30 min, then run at 21 V for 45 min, placed in acetate
solution for 30 min, transferred to 70% ethanol solution for 30
min, dried, stained with Sybr Green for 30 min, then visualized
using an EVOS microscope. TriTek Comet Score freeware was used to
analyze images.
Results
Bone marrow cells harvested from .beta.-globin/GFP transgenic mice
were treated ex vivo with PLGA NPs containing tcPNA1/donor DNA,
tcPNA2/donor DNA and tcPNA3/donor DNA combinations. After 48 hr,
the percentage of GFP+ (corrected) cells was quantified via flow
cytometry, revealing that tcPNA1/donor DNA, tcPNA2/donor and
tcPNA3/donor DNA-containing NPs induced genome modification at
frequencies of .about.1.0%, 0.51% and 0.1% respectively (FIG. 1D).
The higher gene editing activity of tcPNA1 is likely due to its
longer Hoogsteen binding domain, as previously observed
(Schleifman, et al., Chem. Biol. (Cambridge, Mass., U.S.),
18:1189-1198 (2011))). NPs containing the .gamma.-substituted tcPNA
(.gamma.tcPNA4) and donor DNA yielded significantly higher gene
modification (1.62%) (FIG. 1F), showing that the .sup.MP.gamma.
substitutions confer increased biological activity that correlates
with their improved binding properties. NPs with the
.gamma.-substituted but scrambled sequence .gamma.tcPNA4-Scr
produced no modification (FIG. 1F).
Bone marrow cells treated with either blank NPs or NPs containing
.gamma.tcPNA4/donor DNA were plated in methylcellulose medium
supplemented with selected cytokines for growth of
granulocyte/macrophage colonies (CFU-G, CFU-M and CFU-GM) or
combined colonies (CFU-GEMM, granulocyte, erythroid,
monocyte/macrophage, megakaryocyte). The two sets of treated cells
formed myeloid and erythroid colonies at similar frequencies,
indicating that treatment with .gamma.tcPNA4 and donor DNA does not
impair the ability of the progenitor cells to proliferate and
differentiate (FIG. 1G). Sequencing analysis of genomic DNA from
selected GFP-positive methylcellulose colonies confirmed the
presence of the targeted gene modification in the .beta.-globin/GFP
transgene at the IVS2-654 base pair. In other assays for toxicity,
there was no increase in DNA double-strand breaks (DSBs) in the
cells treated with .gamma.tcPNA4/donor DNA-containing NPs compared
to blank NPs based on a single-cell gel electrophoresis assay
(Comet assay) (FIG. 1H) and there was no induction of the
inflammatory cytokines, TNF-alpha or interleukin-6 (IL-6), in the
treated bone marrow cells, consistent with prior work with NPs
containing standard PNAs (McNeer, et al., Gene Therapy, 20:658-669
(2013); Schleifman, et al., Mol. Ther.--Nucleic Acids, 2:e135
(2013); McNeer, et al., Mol. Ther., 19:172-180 (2011)).
Example 3: Gene Modification is Elevated by .gamma.tcPNAs in CD117+
Hematopoietic Cells
Materials and Methods
Cell Sorting and Flow Cytometry
BD Bioscience kit catalog #558451 (BDImag.TM. Hematopoietic
Progenitor Stem Cell Enrichment Set--DM) was used to isolate CD117
cells. Enrichment for CD117 was confirmed by flow cytometry. CD117+
enriched cells were labeled with CD117-APC (BD Pharmingen.TM.
catalog #558451) antibody. Cells were co-labelled with control IgG
antibody (BD Pharmingen.TM. catalog #555746) for gating purposes.
To quantify GFP expression, after CD117 co-labelling, flow
cytometry was performed using FACScaliburS by resuspending cells in
PBS/1% FBS where green fluorescent cells are measured in the Fl1
channel and APC stained cells are in the Fl4 channel Antibodies for
other markers were Ter119 (BD Pharmingen.TM. catalog #561033) and
CD45 APC (BD Pharmingen.TM. catalog #561018).
Results
Previous work indicated that there might be increased activation of
PNA-mediated DNA repair in certain colony-forming progenitors
(McNeer, et al., Gene Therapy, 20:658-669 (2013)). To test this,
whole bone marrow cells were treated with either blank NPs, NPs
containing tcPNA1/donor DNA, or NPs containing .gamma.tcPNA4/donor
DNA. Two days later, flow cytometry was performed to assess the
frequency of GFP+ cells within selected sub-populations.
Substantially elevated gene editing was observed in CD117+ cells
compared to the total CD45+ cell population (FIG. 2A), with a
frequency of 8.6% in CD117+ cells after a single treatment with the
.gamma.tcPNA4/donor DNA NPs. The less potent tcPNA1/donor DNA NPs
still yielded an elevated correction frequency of 2.1% in the
CD117+ cells. The Ter119+ population, which includes more mature
cells committed to the erythroid lineage, showed minimal
susceptibility to gene editing with either PNA.
Next, the predisposition of CD117+ cells to increased gene editing
was tested by first sorting for CD117+ cells prior to treatment
with the NPs (FIG. 2B). An elevated percentage of modification
(7.2%) was again seen in the CD117+ cells after a single treatment
(FIG. 2B).
Example 4: The c-Kit Pathway Mediates Increased Gene Modification
in CD117+ Cells
CD117 (also known as mast/stem cell growth factor receptor or
proto-oncogene c-Kit protein) is a receptor tyrosine kinase
expressed on the surface of hematopoietic stem and progenitor cells
as well as other cell types. Stem cell factor (SCF), the ligand for
c-Kit, causes dimerization of the receptor and activates its
tyrosine kinase activity to trigger downstream signaling pathways
that can impact survival, proliferation, and differentiation.
To explore the mechanism of the increased gene editing in CD117+
cells, the requirement of c-Kit-dependent signaling for elevated
gene correction or whether CD117 simply serves as a marker for the
increased susceptibility to gene editing was distinguished. To do
this, .gamma.tcPNA4/donor DNA NP-mediated gene editing was assayed
in pre-sorted CD117+ cells in the presence or absence of selected
kinase inhibitors (FIG. 2D). Dasatinib, which inhibits the c-Kit
kinase in addition to the BCR/Abl and Src kinases, reduced the gene
editing from 7% to 2.0%. Inhibitors of signaling factors downstream
of c-Kit, including mitogen/extracellular signal-regulated kinase
(MEK) (Binimetinib; MEK162) and phosphatidylinositol-3-kinase
(PI3K) (BKM120), also decreased the gene editing frequencies in
CD117+ cells to 2.6% and 4.1%, respectively (FIG. 2D).
On the other hand, when the CD117+ cells were treated with the
c-Kit ligand, SCF, a significant increase in .gamma.tcPNA4/donor
DNA-mediated gene editing (up to almost 15%) was observed (FIG.
2C). These results indicate that the SCF/c-Kit signaling can
enhance gene editing and identify SCF as a potential agent to
stimulate PNA-mediated gene editing.
Example 5: Expression of DNA Repair Genes are Increased Upon
Activation of the c-Kit+ Pathway
Materials and Methods
Microarray Analysis
Microarray analyses were performed on CD117+ and CD117-cells
obtained from bone marrow of three separate .beta.-globin/GFP mice
at Yale Center of genomic analysis at Yale west campus. Each
replicate cell sample was obtained from a separate mouse. RNA was
extracted from 2.times.10.sup.6 for each sample using the RNeasy
Mini Plus kit from Qiagen, as per the manufacturer's protocol.
Following DNase treatment, total RNA was sequenced and analyzed at
the Yale Center for Genome Analysis. Heat maps were generated using
variance stabilizing transformations of the count data on the basis
of a parametric fit to the overall mean dispersions.
RT-PCR Analysis
Cells were harvested, pelleted, and stored frozen in RNA
stabilization reagent (Qiagen), until ready for RNA extraction. RNA
was extracted from the cell pellets using the RNAeasy Mini Plus kit
from Qiagen, as per the manufacturer's protocol. The Invitrogen
SuperScript III kit was used to generate cDNA from the RNA, as per
the manufacturer's protocol, using 500 ng of RNA per reaction. PCR
reactions contained cDNA, 20% Betaine, 0.2 mM dNTPS, Advantage 2
Polymerase Mix, 0.2 .mu.M of each primer, 2% Platinum Taq, and
Brilliant SYBR Green. Primers and ROX reference dye were obtained
from Stratagene and analysis was conducted using a Mx3000p realtime
cycler. Cycler conditions were 94.degree. C. for 2 min, 40 cycles
of 94.degree. C. 30 s/50.degree. C. 30 s/72.degree. C. 1 min, then
95.degree. C. 1 min. Relative expression were calculated using the
2.DELTA..DELTA.Ct method (Ct<36) and then normalized. Mouse
BRCA2 primers were designed using Primer3 database: BRCA2-3F: 5'
GTTCATAACCGTGGGGCTTA (SEQ ID NO:203) and BRCA2-3R: 5'
TTGGGAAATTTTTAAGGCGA (SEQ ID NO:176). For BRCA2 data analysis GAPDH
were used as control using following primers: 5'-TGATGACATC
AAGAAGGTGGTGAAG-3' (SEQ ID NO:177) and 5'-TCCTTGGAGG
CCATGTGGGCCAT-3' (SEQ ID NO:178). For RAD51 analysis, Rad51 mRNA
was quantified by using TaqMan.RTM. Gene Expression Assay (Life
technologies, Mm00487905_m1) kit and using gene 18S (Life
technologies, Mm03928990_g1) as a control.
Western Blot Analysis
CD117+ and CD117-cells were isolated from .beta.-globin/GFP mice
and protein was extracted with Radio-Immunoprecipitation Assay
(RIPA) lysis buffer. 50-100 .mu.g total protein was run on SDS/PAGE
gels and transferred to nitrocellulose membranes. Antibodies used
were: Anti-BRCA2 (Ab-1) mouse mAb (EMD Millipore, OP95-100 ug)
anti-RAD51-antibody (Santa Cruz biotechnology, SC 8349)).
Results
The increased gene editing in the c-Kit+(CD117) cells was not
explained by differential uptake of the NPs, as there were no
detectable differences in uptake across several bone marrow cell
sub-populations. Gene expression patterns in the c-Kit+ cells were
evaluated for increased DNA repair gene expression. Gene expression
analyses were performed on sorted CD117+ and CD117-cells from whole
bone marrow from the .beta.-globin/GFP mice using Illumina
arrays.
TABLE-US-00015 TABLE 3 Selected genes that were up-regulated in
CD117+ enriched cells as compared to CD117- cells with increased
expression of transcripts expected to be associated with CD117
including c-Kit, VEGF (vascular endothelial growth factor), Sca1
(stem cell antigen-1), and Erdr1 (erythroid differentiation
regulator 1). Fold Change CD117 CD117 CD117 negative/ Gene Negative
Positive CD117 positive P value c-Kit 593.98 2368.32 -3.98715
0.0051 VEGF 344.34 1109.97 -3.22341 0.0084 Sca1 208.24 490.86
-2.35711 0.0126 Erdr1 1011.81 2760.26 2.72805 0.0319
Numerous genes involved in DNA repair, including BRCA1, BRCA2,
Rad51, ERCC2, XRCC2, XRCC3, showed higher levels of expression in
CD117+ cells. Two key HDR genes expected to play a role in
PNA-induced recombination, BRCA2 and Rad51, were among the
upregulated genes detected by the array. Increased expression of
these genes was confirmed in CD117+ cells at the mRNA level by
quantitative RT-PCR (FIGS. 2E and 2F) and at the protein level by
western blot.
Based on these findings, activation of the c-Kit pathway by SCF
treatment to further increase DNA repair gene expression was
examined Gene expression profiling on SCF-treated CD117+ cells
versus untreated CD117+ cells showed additional up-regulation of
numerous DNA repair genes (FIG. 2G), again including Rad51 and
BRCA2.
Example 6: The c-Kit Pathway Induces Functionally Elevated DNA
Repair
Materials and Methods
Reporter Gene Assay for Homology-Dependent Repair
An inactivating I-Sce1 site was cloned 56 amino acids into the
firefly luciferase open reading frame under the control of a CMV
promoter. The reporter construct also contains a promoterless
luciferase gene used as a template for homologous recombination. A
double-strand break in the luciferase reporter is created by in
vitro digestion with the I-Sce I restriction enzyme (NEB #R0694L).
Plasmid DNA was digested with I-Sce 1 for 1 hour at 37.degree. C.
at a ratio of 10 units enzyme to 1 .mu.g DNA and then the enzyme
was inactivated at 65.degree. C. for 20 minutes. The linearization
of the plasmid was confirmed for each digestion via gel
electrophoresis and the linear plasmid was purified using the
Qiagen Qiaquick spin columns. After separation CD117+ and
CD117-cells from bone marrow of .beta.-globin/GFP transgenic mice,
cells were transfected using the Lonza 2b Nucleofector Device.
5.times.10.sup.5 cells were transfected with 1 .mu.g of either the
luciferase reporter vector or a positive control firefly luciferase
expression vector, along with 50 ng of a renilla luciferase
expression plasmid as a transfection efficiency control. All
transfections were performed in triplicate. After transfection the
cells were plated at a density of 5.times.10.sup.5 cells/ml in
12-well plates. After 24 hours incubation post transfection,
luciferase activity was measured using the Promega Dual Luciferase
Assay Kit. In each sample firefly luciferase activity was
normalized to the renilla luciferase transfection control. Reporter
reactivation was calculated as a ratio of normalized firefly
luciferase activity in the cells transfected with the reporter
plasmid to the positive control.
Results
To test whether the above increases in DNA repair gene expression
could be correlated with functional differences in DNA repair, a
luciferase-based assay was used to quantify repair of DNA
double-strand breaks (DSBs) by HDR. In this assay, repair of a DSB
in a reporter plasmid via intramolecular homologous recombination
creates ("reactivates") a functional luciferase gene (FIG. 2H), and
so the assay provides a measure of HDR capacity (FIG. 2J). The
results show increased luciferase reactivation in CD117+ compared
to CD117-cells (FIG. 2H). The repair activity in the CD117+ cells
was diminished by treatment with the kinase inhibitors MEK162,
BKM120 and dasatnib (FIG. 2H); conversely, it was further boosted
by SCF treatment (FIG. 2I). These results indicate that a
functional c-Kit signaling pathway mediates increased HDR.
Example 7: In Vivo Gene Editing by Intravenous Injections of
PNA/DNA NPs is Enhanced by SCF Treatment
Materials and Methods
Mouse Models and In Vivo Treatments
All animal use was in accordance with the guidelines of the Animal
Care and Use Committee of Yale University and conformed to the
recommendations in the Guide for the Care and Use of Laboratory
Animals (Institute of Laboratory Animal Resources, National
Research Council, National Academy of Sciences, 1996).
The .beta.-globin/GFP transgenic mice were obtained from Ryszard
Kole, University of North Carolina (Sazani, et al., Nat.
Biotechnol., 20:1228-1233 (2002)). For treatment of the mice, where
indicated SCF (15.6 ug per mouse, Recombinant Mouse SCF,
carrier-free, R&D catalog #455-mc-050/CF) was injected
intraperitoneally 3 hrs prior to treatment with 4 mg of NPs in 150
.mu.l PBS delivered via retro-orbital intravenous injection. In
some cases, mice were sacrificed 48 hrs after the NP injections and
bone marrow and spleen cells were harvested for further analysis.
The bone marrow and spleen cells (500,000 each) were co-labelled
with APC conjugated antibodies as described above and flow
cytometry was performed as above. For deep sequencing analyses,
CD117+ cells were isolated based on magnetic separation methods
according to BD Bioscience protocol (BDImag.TM. Hematopoietic
Progenitor Stem Cell Enrichment Set--DM), and genomic DNA from
three mice was pooled followed by sequence analysis as described
(McNeer, et al., Gene Therapy, 20:658-669 (2013)).
The IVS2-654 .beta.-thalassemic mice were also obtained from
Ryszard Kole, University of North Carolina (Svasti, et al., Proc
Natl Acad Sci USA, 106:1205-1210 (2009)). For treatment of the
mice, where indicated SCF (15.6 ug per mouse, Recombinant Mouse
SCF, carrier-free, R&D catalog #455-mc-050/CF) was injected
intraperitoneally 3 hrs prior to treatment with 4 mg of NPs in 150
.mu.l PBS delivered via retro-orbital intravenous injection. Each
mouse received 4 treatments given at 48 hr intervals. Mice were
anesthetized with isoflurane followed by retro-orbital bleeding
(.about.100 .mu.L) using ethylenediaminetetraacetic acid-treated
glass capillary tubes. The blood was evacuated into tubes with 5
.mu.L of 0.5 M EDTA acid in heparinized coated tubes. Complete
blood counts were performed using a Hemavet 950FS (Drew Scientific,
Oxford, Conn.) according to the manufacturer's protocol. Slides
containing blood smears were stained with Wright and Giemsa stain
for microscopy. Methylene blue staining was used for reticulocyte
counts. Spleen images and weights were taken after selected mice
were sacrificed on day 36 after the last treatment. Harvested
spleens were fixed in 10% neutral buffered formalin and processed
by Yale Pathology Tissue Services for H&E, CD61 and E cadherin
staining.
For assigning animals into treatment groups as listed above,
littermate animals were genotyped, and then the pups carrying the
required genotypes (either .beta.-globin/GFP transgenic mice or
IVS2-654 .beta.-thalassemic mice) were randomized into the several
treatment groups in cohorts of 3 to 6, as indicated. The
investigators were not blinded as to treatment groups.
Genomic DNA Extraction and Deep Sequence Analysis
Genomic DNA from mouse cells treated ex vivo or in vivo, as
indicated, was harvested using the Wizard Genomic Purification Kit
(Promega), and then electrophoresed in a 1% low melting point
agarose gel in TAE, to separate genomic DNA from possible residual
PNA and/or DNA oligonucleotide. The high-molecular weight species,
representing genomic DNA, was cut from the agarose gel and
extracted using the Wizard SV Gel and PCR Clean-Up System (Promega)
according to manufacturer's instructions. Once genomic DNA was
isolated from treated cells or mouse tissue, PCR reactions were
performed with high fidelity TAQ polymerase. Each PCR tube
consisted of 28.2 .mu.L dH2O, 5 .mu.L 10.times.HiFi Buffer, 3 .mu.L
50 mM MgCl2, 1 .mu.L DNTP, 1 .mu.L each of forward and reverse
primer, 0.8 .mu.L High Fidelity Platinum Taq Polymerase
(Invitrogen, Carlsbad Calif.) and 10 .mu.L DNA template. PCR
products were prepared by end-repair and adapter ligation according
to Illumina protocols (San Diego, Calif.), and samples sequenced by
the Illumina HiSeq with 75 paired-end reads at the Yale Center for
Genome Analysis. Samples were analyzed as previously described
(McNeer, et al., Gene Therapy, 20:658-669 (2013)). Primers for deep
sequencing were designed using Primer3 data base. The primers used
for .beta.-globin intron 2 were as follows: forward primer: 5'
TATCATGCCTCTTTGCACCA (SEQ ID NO:179); reverse primer: 5'
AGCAATATGAAACCTCTTACATCA (SEQ ID NO:180). Primers for off-target
sites of partial homology were as follows; forward primer is listed
first:
TABLE-US-00016 Vascular cell adhesion protein precursor 1 (5'
AGATAATTATTGCCTCCCACTGC (SEQ ID NO: 181) and 5'
AATGGAAGGGCATGCAGTCA (SEQ ID NO: 182)); Polypyrimidine tract
binding protein (5' CCCAATCCTGAATCCTGGCT (SEQ ID NO: 183) and 5'
CATACTGATGTCTGTGGCTTGA (SEQ ID NO: 184)); Protocadherin fat 4
precursor (5' AAGCTCAAACCTACCAGACCA (SEQ ID NO: 185) and 5'
AGCTGGAAGCTTCTTCAGTCA (SEQ ID NO: 186)); Olfactory receptor 266 (5'
CCCTCTGTGGACTGAGGAAG (SEQ ID NO: 187) and 5' TGATGAGCTACGGGTATGTGA
(SEQ ID NO: 188)); Syntaxin binding protein (5'
CAAAAAGCCTTAAGCAAACACTC (SEQ ID NO: 189) and 5'
TCTCTCCCTCAGCATCTATTCC (SEQ ID NO: 190)); Muscleblind like protein
(5' TGTGTTTGTTTATGGATACTTGAGC (SEQ ID NO: 191) and 5'
GCATGCACAATAAAGGCACT (SEQ ID NO: 192)); Ceruloplasmin isoform (5'
CATGGGAAACAGTCAAAAGAAA (SEQ ID NO: 193) and 5' TGTAGGTTTCCCCACAGCTT
(SEQ ID NO: 194)).
Results
The potential for in vivo gene editing in the .beta.-globin/GFP
transgenic mice was explored by intravenous injection of NPs
containing .gamma.tcPNA4 and donor DNA. The ability of SCF
treatment to enhance gene editing in vivo was also tested. Mice
were treated with a single intravenous dose of 4 mg NPs in 150
.mu.l PBS, and 2 days later the mice were sacrificed for analysis
of gene editing in cells from the bone marrow and spleen. Some mice
also received murine SCF (15.6.mu.g) given by intraperitoneal
injection 3 hr prior to the NP injection, as indicated. In vivo
gene editing was scored by GFP expression in marker-sorted cell
populations from bone marrow and spleen (FIGS. 3A and B). The
highest levels of gene editing were seen in CD117+ cells from bone
marrow and spleen of the SCF-treated mice, with frequencies in the
range of 1% in several mice, and average frequencies in the 0.4% to
0.5% range.
These results were confirmed by performing deep sequencing analysis
on genomic DNA from CD117+ cells isolated from bone marrow and
spleen of treated mice (FIG. 3C), which revealed gene editing
frequencies in the range of 0.2% in the bone marrow of mice treated
with NPs alone and 0.6% in mice receiving SCF along with the NPs,
consistent with the frequencies of gene correction quantified by
GFP expression. Deep-sequencing was also used to assess off-target
effects in the bone marrow cells of the mice that were treated with
SCF and .gamma.tcPNA4 and donor DNA NPs (Table 4). By BLAST
analysis, seven off-target sites with partial homology to the
target site of .gamma.tcPNA4 in .beta.-globin intron 2 were
identified. Mutation frequencies at these sites were quantified via
deep sequencing. Extremely low frequencies of off-target effects
were found in the .gamma.tcPNA4/donor DNA treated mice, with six
sites showing no detectable sequence changes out of millions of
reads and two sites showing modification frequencies of only
0.0074% and 0.00018% compared to 0.56% at the targeted
.beta.-globin site. (Table 4). The overall off-target modification
frequency at all seven sites combined was 0.00034%, 1,647-fold
lower than the frequency of the targeted gene editing.
TABLE-US-00017 TABLE 4 Off-target effects in bone marrow cells
following intravenous treatment of .beta.-globin/GFP mice with
.gamma.tcPNA4/donor DNA NPs. Sequences of partial Size of region
Alleles Number Gene locus homology (5' to 3') sequenced sequenced
modified Frequency % .beta.-globin TGCCCTGAAAGAAA 128 1399786 78833
0.56 GAGA (SEQ ID NO: 195) Vascular cell AGCCCTGAAAGAAA 111 480013
0 0 adhesion protein GAGA (SEQ ID precursor 1 NO: 196)
Polypyrimidine GAACCTGAAAGAAA 101 349723 26 0.0074 tract binding
GAGA (SEQ ID protein NO: 197) Protocadherin fat 4 CACCCTGAAAGAAA
115 73245 0 0 precursor GAAA (SEQ ID NO: 198) Olfactory receptor
AAGCCTGAAAGAAA 172 1092990 2 0.00018 266 GAGT (SEQ ID NO: 199)
Syntaxin binding AGAAATGAAAGAAA 150 2478636 0 0 protein GAGA (SEQ
ID NO: 200) Muscleblind like GGTGGTGAAAGAAA 165 2331971 0 0 protein
GAGA (SEQ ID NO: 201) Ceruloplasmin AGGACTGAAAGAAA 154 1390439 0 0
isoform GAGT (SEQ ID NO: 202) Total off-target 8197017 28
0.00034
The top seven gene loci with partial homology to the 18 bp
.gamma.tcPNA4 target site in .beta.-globin intron 2 were
identified, with the sequences as indicated. .beta.-globin/GFP mice
were treated with SCF followed by intravenous infusion with NPs
containing .gamma.tcPNA4/donor DNA, and genomic DNA from c-Kit+
bone marrow cells was subject to deep sequencing analysis at these
loci. The size of the region sequenced around each site is listed,
along with the number of alleles sequenced and the number of
alleles with modified sequences.
Example 8: SCF and PNA NP Treatment can Correct a Genomic Mutation
in a Mouse .beta.-Thalassemia Disease Model
To test the extent to which combined SCF and PNA NP treatment in
vivo could correct a human .beta.-thalassemia mutation in a mouse
disease model, a transgenic mouse line was utilized in which the
two (cis) murine adult beta globin genes were replaced with a
single copy of the human .beta.-globin gene with the
thalassemia-associated IVS2-654 mutation (Svasti, et al., Proc Natl
Acad Sci USA, 106:1205-1210 (2009)). Homozygous mice do not
survive, and heterozygotes have a moderate form of
.beta.-thalassemia, with marked hemolytic anemia, microcytosis, and
increased MCHC and red cell distribution width reflecting reduced
amounts of mouse .beta.-globin and no human .beta.-globin (Lewis,
et al., Blood, 91:2152-2156 (1998); Svasti, et al., Proc Natl Acad
Sci USA, 106:1205-1210 (2009)). Blood smears from these mice show
erythrocyte morphologies consistent with .beta.-thalassemia.
Treatment groups for this experiment included (1) blank NPs; (2)
SCF treatment alone (no NPs); (3) SCF plus .gamma.tcPNA4/donor DNA
NPs; and (4) SCF plus .gamma.tcPNA4-Scr/donor DNA. SCF injections
were given i.p., and NPs were given i.v. via retro-orbital
injection. Each treatment group consisted of six mice, and each
mouse received four treatments at two-day intervals. Blood smears
examined at day 0 (before treatment) and at day 36 after the last
treatment showed marked improvement in RBC morphology on day 36 in
the .gamma.tcPNA4/donor DNA treated mice but not in the mice
treated with either blank NPs, SCF alone, or SCF plus
.gamma.tcPNA4-Scr/donor DNA. Compared to wild-type, the untreated
group (and corresponding control animals) exhibit extreme
poikilocytosis which is typical of .beta.-thalassemia, as well as
the presence of numerous target cells, cabot rings, anisochromasia,
and ovalocytosis, changes characteristic of .beta.-thalassemia.
Treatment with .gamma.tcPNA4/donor DNA and SCF ameliorates the
poikilocytosis and yields a reduction in anisocytosis,
ovalocytosis, and target cells indicative of reduced alpha-globin
precipitation in the RBCs.
CBC analyses performed on blood samples taken at 30, 45, 60, and 75
days post-treatment from mice in each group showed persistent
correction of the anemia in the mice treated with SCF plus the
.gamma.tcPNA4/donor DNA NPs (FIG. 4A-4C), with elevation of the
blood hemoglobin levels into the normal range. Only the SCF plus
.gamma.tcPNA4/donor DNA-treated mice achieved and maintained
hemoglobin levels within the normal range during the duration of
the experiment, reflecting the increased hemoglobin stability
conferred by the gene editing.
The anemia was not improved in any of the controls. Reticulocyte
counts were observed in mice treated with SCF plus the
.gamma.tcPNA4/donor DNA NPs but not in the mice treated with blank
NPs (FIG. 4D). Deep sequencing analyses were performed on genomic
DNA extracted from bone marrow cells of three mice from each group
that were sacrificed on day 36 post-treatment. Correction of the
targeted mutation was seen at a frequency of almost 4% in the
.gamma.tcPNA4/donor DNA treated group (FIG. 4E), whereas no
correction was seen in the mice treated with blank NPs. In
addition, in keeping with the correction of the anemia and
suppression of the reticulocytosis, the .gamma.tcPNA4/donor DNA
treated mice also showed reduced splenomegaly at 36 days
post-treatment.
Consistent with the reduced splenomegaly, histologic examination of
the spleens of mice sacrificed on day 36 showed substantially
improved splenic architecture specifically in the
.gamma.tcPNA4/donor DNA treated mice. The regular splenic
histologic pattern of white pulp (lymphoid follicles) surrounded by
rims of red pulp as seen in the wild-type spleen is disrupted in
the .beta.-thalassemic animals (blank NPs, SCF alone, SCF plus
scrambled .gamma.tcPNA4-Scr/donor DNA NPs) due to extramedullary
hematopoiesis, which results in an expansion in the red pulp
(causing the splenomegaly) and disruption of the white pulp. The
CD61 and Ecad immunohistochemical stains highlight the increased
cellularity characteristic of extramedullary hematopoiesis and
demonstrate that the expanded red pulp in the .beta.-thalassemic
animals includes elevated numbers of megakaryocytes and erythroid
precursors, respectively. This increased cellularity is
substantially ameliorated in the .gamma.tcPNA4/donor DNA treated
mice.
Deep-sequencing was also used to assess off-target effects in the
bone marrow of the in vivo treated thalassemic mice. As above,
seven off-target sites with partial homology to the binding site of
.gamma.tcPNA4 in the .beta.-globin gene were analyzed. Only
extremely low frequencies of off-target effects were found in the
.gamma.tcPNA4/donor DNA-treated thalassemic mice (Table 5), similar
to the results in the .beta.-globin/GFP transgenic mice (Table 4).
The overall off-target modification frequency in this case was
0.0032%, 1,218-fold lower than the frequency off .beta.-globin gene
editing.
TABLE-US-00018 TABLE 5 Off-target effects in bone marrow cells
following intravenous treatment of .beta.-thalassemic mice with SCF
and .gamma.tcPNA4/donor DNA NPs. Size of Sequences of partial
region Alleles Number Gene locus homology (5' to 3') sequenced
sequenced modified Frequency % .beta.-globin TGCCCTGAAAGAAA 128
8615313 337192 3.9 GAGA (SEQ ID NO: 195) Vascular cell
AGCCCTGAAAGAAA 111 482051 0 0 adhesion protein GAGA (SEQ ID
precursor 1 NO: 196) Polypyrimidine GAACCTGAAAGAAA 101 355567 2
.00056 tract binding GAGA (SEQ ID protein NO: 197) Protocadherin
fat CACCCTGAAAGAAA 115 123158 0 0 4 precursor GAAA (SEQ ID NO: 198)
Olfactory AAGCCTGAAAGAAA 172 1099880 262 0.0231 receptor GAGT (SEQ
ID 266 NO: 199) Syntaxin binding AGAAATGAAAGAAA 150 2493024 0 0
protein GAGA (SEQ ID NO: 200) Muscleblind like GGTGGTGAAAGAAA 165
2336715 0 0 protein GAGA (SEQ ID NO: 201) Ceruloplasmin
AGGACTGAAAGAAA 154 1397271 0 0 isoform GAGT (SEQ ID NO: 202) Total
off-target 8287666 268 .0032
The top seven gene loci with partial homology to the 18 bp
.gamma.tcPNA4 target site in .beta.-globin intron 2 were
identified, with the sequences as indicated. Thalassemic mice were
treated with SCF followed by intravenous infusion with NPs
containing .gamma.tcPNA4/donor DNA, and genomic DNA from c-Kit+
bone marrow cells was subject to deep sequencing analysis at these
loci. The size of the region sequenced around each site is listed,
along with the number of alleles sequenced and the number of
alleles with modified sequences.
In sum the results above demonstrate that chemically modified
.gamma.PNAs and donor DNAs delivered intravenously via polymer NPs,
and given in combination with SCF treatment, can mediate gene
editing in vivo at a level sufficient to ameliorate the disease
phenotype in the thalassemic mice. Sustained reversal of the
anemia, with normalization of serum hemoglobin concentrations and
suppression of the reticulocytosis were induced. A morphologic
improvement in RBC cytology, indicative of improved RBC stability,
along with reduced extramedullary hematopoiesis and reduction in
splenomegaly were observed. This constellation of findings
indicates that the disclosed therapeutic approach has the potential
to deliver a substantial clinical response that would relieve the
morbidity and mortality associated with .beta.-thalassemia.
There are at least two important advances for gene editing in this
work. One advance is the incorporation of next generation PNA
chemistry by substitution within the polyamide backbone at the
gamma position to consistently yield increases in gene editing
frequencies compared to standard PNAs. This increased efficacy
correlates with the enhanced DNA binding properties of .gamma.PNAs,
which take on a pre-organized helical conformation enforced by the
miniPEG .gamma. substitution.
Another advance is the finding that the SCF/c-Kit pathway promotes
increased gene editing by triplex-forming PNAs and donor DNAs. Upon
ex vivo treatment of bone marrow cells with .gamma.PNAs, the gene
editing frequency in c-Kit+ cells was as high as 8%. The
combination of SCF treatment with the .gamma.PNAs yielded even
higher frequencies in the c-Kit+ cells, with just over 15% in a
single treatment. In vivo, treatment of transgenic mice carrying a
.beta.-globin/GFP reporter transgene by i.p. injection of SCF
followed by intravenous administration of NPs containing
.gamma.PNAs and donor DNAs yielded gene editing in CD117+ cells in
the bone marrow and spleen at frequencies up to 1% in a single
treatment. Prompted by these results in reporter mice, gene editing
was tested in the thalassemic mouse model via simple intravenous
injection of the optimized combination of SCF and .gamma.PNA/donor
DNA NPs given four times at two-day intervals. This regimen yielded
gene editing at a frequency of almost 4% in total bone marrow cells
and produced sustained amelioration of the disease phenotype,
achieved in a minimally invasive manner without the need for stem
cell harvest or transplantation.
Importantly, in a series of ex vivo and in vivo assays for
hematopoietic colony formation, for induction of inflammatory
cytokines, for generation of strand breaks, and for off-target
mutagenesis by deep sequencing, there was essentially no measurable
cellular toxicity and very low off-target genome effects from the
.gamma.PNA-containing NPs, providing a possible safety advantage
relative to other gene editing approaches (Cradick, et al., Nucleic
Acids Res., 41:9584-9592 (2013)).
CD117 is the product of the c-Kit gene and is a receptor tyrosine
kinase that mediates downstream signalling to multiple cellular
pathways. The results discussed above indicate that activation of
this pathway promotes gene editing, rather than CD117 simply being
a marker for the phenotype. Inhibition of the c-Kit kinase with
dasatinib reduces the frequency by almost 4-fold, whereas treatment
with SCF almost doubles the frequency. Mechanistically, CD117+ bone
marrow cells, in comparison to CD117-cells, have elevated levels of
expression of numerous DNA repair genes, including factors in the
HDR pathway that prior work has shown is required for
triplex-induced gene editing (Vasquez, et al., Science, 290:530-533
(2000); Rogers, et al., Proc. Natl. Acad. Sci. USA, 99:16695-16700
(2002); Datta, et al., J Biol Chem, 276:18018-18023 (2001);
Vasquez, et al., Proc Natl Acad Sci USA, 99:5848-5853 (2002)). When
CD117+ cells are treated with SCF, expression of these DNA repair
genes is increased even more, correlating with a further increase
in gene editing.
In addition, the results show that the elevated expression of DNA
repair genes in CD117+ cells is associated with functionally
increased HDR activity using an assay for recombination between
reporter gene constructs. Treatment of the CD117+ cells with SCF
produced a further 2-fold increase in HDR, whereas dasatinib and
the other inhibitors yielded reductions in HDR activity. These
results show the functional importance of the c-Kit pathway in
promoting HDR and provide further mechanistic insight into gene
editing pathways.
The 4% frequency of bone marrow gene editing achieved in the
thalassemic mice was sufficient to achieve a clear improvement in
phenotype, with blood hemoglobin levels rising into the normal
range, suppression of the reticulocytosis, and reduction in the
splenomegaly that is otherwise associated with extramedullary
hematopoiesis. The observation that gene correction at a frequency
of 4% could confer a phenotypic impact is consistent with
transplantation studies in thalassemic mice and in patients in
which mixed chimerism at one ratio of wild-type donor to
thalassemic recipient cells in the marrow has produced much higher
proportions of donor RBCs in the periphery (Andreani, et al., Bone
Marrow Transplant, 7(Suppl 2):75 (1991); Felfly, et al., Mol Ther,
15:1701-1709 (2007)). This effect has been attributed to increased
survival and enrichment of genetically corrected erythroblasts
during erythropoiesis, decreased ineffective erythropoiesis, and
increased survival in the circulation of corrected erythrocytes
relative to thalassemic RBCs (Miccio, et al., Proc Natl Acad Sci
USA, 105:10547-10552 (2008)).
Overall, these results support the feasibility of NP-mediated
delivery of .gamma.PNAs and donor DNAs as a therapeutic strategy to
achieve in vivo gene editing for treatment of human genetic
disorders. The results described above demonstrate effective
NP-mediated gene editing in bone marrow, but other recent work has
shown that NP delivery to lung airway epithelia is also possible as
a potential means to achieve correction of the CFTR gene mutation
associated with cystic fibrosis (Fields, et al., Adv Healthc Mater
(2014); McNeer, et al., Nature Communications in press (2015)).
The finding that SCF stimulates gene editing identifies SCF as a
possible pharmacologic means to boost gene editing, a strategy that
may be applicable not just to PNA-mediated gene editing as shown
here but possibly also to editing by other methods, such as
CRISPR/Cas9, SFHR, or ZFNs. Furthermore, even though the
.gamma.PNAs show consistently improved gene editing potency, the
level of off-target effects in the genome remains extremely low.
This is in keeping with the lack of any intrinsic nuclease activity
in the PNAs (in contrast to ZFNs or CRISPR/Cas9), and reflects the
mechanism of triplex-induced gene editing, which acts by creating
an altered helix at the target-binding site that engages endogenous
high fidelity DNA repair pathways. The SCF/c-Kit pathway also
stimulates these same pathways, providing for enhanced gene editing
without increasing off-target risk or cellular toxicity.
Example 9: Repair Proteins Modulate Triplex-Forming PNA Mediated
Gene Editing
Materials and Methods
Skin fibroblasts were isolated from the .beta.-globin/GFP mice
(intron 2 of human .beta.-globin inserted with in the GFP coding
regions) and grown in culture in DMEM medium plus 10% FCS. The
intron contains the IVS2-654 (C.fwdarw.T) mutation. The gene
correction assay is illustrated in FIG. 5A.
The fibroblasts were treated ex vivo with nanoparticles containing
tcPNA1+ Donor DNA and 72 hours later flow cytometry analysis was
performed to quantify the % gene correction based on the frequency
of GFP positive cells. In some cases, DNA repair inhibitors or
other small molecule inhibitors were given 48 hours before the
nanoparticle treatment.
TABLE-US-00019 tcPNA1: (SEQ ID NO: 35)
H-KKK-TTTJTJJ-OOO-CCTCTTTGCACCATTCT-KKK-NH2 Donor DNA: (SEQ ID NO:
175) 5'A(s)A(s)A(s)GAATAACAGTGATAATTTCTGGGTTAAGGCAAT
AGCAATATCTCTGCATATAAA(s)T(s)A(s)T 3'
TABLE-US-00020 TABLE 6 ATR pathway inhibitors Working Drug Inhibits
Concentration MIRIN Mre11 20 .mu.M KU55933 ATM 20 .mu.M VE-821 ATR
10 .mu.M NU7441 DNAPKcs 20 .mu.M LCA Polymerase .beta. 50 .mu.M
L189 DNA ligase I III IV 50 .mu.M
TABLE-US-00021 TABLE 7 CHK1, DNA polymerase alpha, and polyADP
ribose polymerase inhibitors Working Drug Inhibits Concentration
Aphidicolin Polymerase .alpha. 1 .mu.g/ml SB218075 Chk1 1 .mu.M AZD
PARP 20 .mu.M
Results
Inhibition of ATR boosts gene editing in the GFP/beta globin gene
correction assay in mouse fibroblasts. The results are presented in
FIG. 5B.
Inhibition of CHK1 substantially boosts gene editing in GFP/beta
globin gene correction assay Inhibition of DNA polymerase alpha (by
aphidicolin) or of polyADP ribose polymerase by AZD-2281 (olaparib)
also boosts gene editing. The results are presented in FIG. 5C.
Inhibition of heat shock protein 90 (HSP90) by STA-9090/Ganetespib
enhances gene editing in the GFP/beta globin gene correction assay.
The results are presented in FIG. 5D.
Example 10: Partial .gamma. Substitution in the Hoogsteen Domain
Increases Gene Correction Efficiency
Materials and Methods
TABLE-US-00022 (SEQ ID NO: 93)
lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys- lys-lys (SEQ ID NO:
69) lys-lys-lys-TJTJJTTT-OOO-TTTCCTCTATGGGTAAG-lys- lys-lys
Results
A substantial increase in gene editing in F508del CFTR using
.gamma.tcPNAs with just partial .gamma. substitution and only in
the Hoogsteen domain of CF PNA2. As shown in FIGS. 7A and 7B, with
only 4 .gamma. residues in the Hoogsteen domain, a more than 50%
increase in activity for CFTR gene correction was achieved in CFBE
cells (via NPs containing .gamma.tcPNAs) as judged by the MQAE
assay (FIG. 7A). A substantial increase in activity with
.gamma.tcPNA containing NPs was also achieved in vivo in CF mice
following intranasal delivery, as determined by NPD measurements
(FIG. 7B).
Example 11: Nanoparticle Delivered tcPNA and Donor Oligonucleotide
Correct a Sickle Cell Mutation In Vivo
Materials and Methods
PNAs
TABLE-US-00023 SCD-tcPNA 1: (SEQ ID NO: 59)
H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-KKK-NH.sub.2 SCD-tcPNA 2: (SEQ
ID NO: 213) H-KKK-TTJJTJT-OOO-TCTCCTTAAACCTGTCTT-KKK-NH.sub.2
SCD-tcPNA 3: (SEQ ID NO: 60)
H-KKK-TJTJTTJT-OOO-TCTTCTCTGTCTCCACAT-KKK-NH.sub.2.
K indicates lysine; J, pseudoisocytosine (for C) for pH-independent
triplex formation. O,8-amino-2,6,10-trioxaoctanoic acid linkers
connecting the Hoogsteen and Watson-Crick domains of the
tcPNAs.
Donor
TABLE-US-00024 (SEQ ID NO: 161)
5'-T(s)T(s)G(s)CCCCACAGGGCAGTAACGGCAGACTTCTCCTCAGG
AGTCAGGTGCACCATGGTGTCTGTT(s)T(s)G(s)-3',
wherein the bolded and underlined residue is the correction and
"(s)" indicates a phosphorothioate internucleoside linkage.
Mouse Models for Sickle Cells Disease
In sickle cell disease (SCD), the mutation (GAG.fwdarw.GTG) at
codon 6 results in glutamic acid changed to valine. For correction
of this SCD mutation site in vivo, in vivo studies were performed
in two mouse models:
(1) sickle cell gene knock in murine model also known as the
Berkeley mouse model introduced by Paszty C, Brion C M, Manci E,
Witkowska H E, Stevens M E, Mohandas N, Rubin E M., "Transgenic
knockout mice with exclusively human sickle hemoglobin and sickle
cell disease." Science. 1997 Oct. 31; 278(5339):876-8. PMID:
9346488 and
(2) the Townes mouse model developed by Ryan.TM., Ciavatta D J,
Townes T M., "Knockout-transgenic mouse model of sickle cell
disease." Science. 1997 Oct. 31; 278(5339):873-6. PMID:
9346487.
Both of these mouse models express exclusively human sickle
hemoglobin (HbS). They were produced by generating transgenic mice
expressing human .alpha.-, .gamma.-, and .beta..sup.s-globin that
were then bred with knockout mice that had deletions of the murine
.alpha.- and .beta.-globin genes. Thus the resulting progeny no
longer express mouse .alpha.- and .beta.-globin. Instead, they
express exclusively human .alpha.- and .beta..sup.s-globin. Hence,
the mice express human sickle hemoglobin and possess many of the
major hematologic and histopathologic features of individuals with
SCD.
Nanoparticles
tcPNAs and donor DNAs, at a molar ratio of 2:1, were incorporated
into PLGA NPs. The NP formulations were evaluated by scanning
electron microscopy (SEM) and dynamic light scattering (DLS).
Treatment Protocol
Three each (i.e., n=3) of Berkley and Townes mice were treated with
(1) Blank PLGA nanoparticles, (2) 4 treatments of sc-tcPNA1/donor
DNA in PLGA nanoparticles, (3) 4 treatments of sc-tcPNA2/donor DNA
in PLGA nanoparticles, (4) 4 treatments of sc-tcPNA3/donor DNA in
PLGA nanoparticles. Mice were injected intravenously with 2 mg of
NPs containing the PNAs and donor DNAs every two days for a total
of 4 injections. After the last treatment, bone marrow and spleen
were collected for histology, deep sequencing, and restriction
enzyme digest.
Results
Three polypurine sites in the .beta.-globin gene in the vicinity of
the SCD codon. Triplex formation can catalyze recombination at
sites up to several hundred base pairs away. A series of tcPNAs
were designed to bind to selected polypurine stretches in the
.beta.-globin gene in the vicinity of the SCD mutation and
synthesized (FIG. 10A). A sense donor DNA (a single-stranded 60-mer
matching nucleotides in .beta.-globin gene and end-protected from
degradation by 3 terminal phosphorothioate internucleoside linkages
was also designed.
More specifically, gel mobility shift assays demonstrated binding
of SCDtcPNA1, SCDtcPNA2, SCDtcPNA3 to 120 bp double-stranded DNA
fragments containing .beta.-globin sequences. Each 120 bp dsDNA
contained the binding site for the respective tcPNAs. The binding
assays revealed that all synthesized SCD tcPNAs bind specifically
to double-stranded genomic DNA under physiological conditions.
Poly (lactic-co-glycolic acid) (PLGA) NPs can effectively deliver
PNA/donor DNA combinations into primary human and mouse
hematopoietic cells with essentially no toxicity. Here, tcPNAs and
donor DNAs, at a molar ratio of 2:1, were incorporated into PLGA
NPs. The NP formulations were evaluated by scanning electron
microscopy (SEM) and dynamic light scattering (DLS). All the NPs
exhibited sizes within the expected range and showed uniform charge
distribution (FIGS. 10B-10C).
Next, correction of SCD mutation in the two disease mouse models
was carried out as described above. Treatment groups included (1)
blank NPs; (2) SCD tcPNA1/donor DNA; (3) SCD tcPNA2/donor DNA; and
(4) SCD tcPNA3/donor DNA. Mice were injected intravenously with 2
mg of NPs containing the PNAs and DNAs every two days for a total
of 4 injections.
Deep sequencing analyses of the human beta globin alleles were
performed on genomic DNA taken from total bone marrow cells of mice
on day 36 post-treatment. Correction of the SCD mutation was seen
at a frequency of almost 1.5% in the SCD tcPNA1/donor DNA treated
group in the Townes mice (FIG. 10D) and 1.2% gene correction in the
Berkley mice (FIG. 10E), whereas no correction was seen in the mice
treated with blank NPs. The results were confirmed using
restriction enzyme (Bsu361) digestion which cuts only when the
sequence at codon 6 has been edited from the SCD mutation to the
wild-type sequence.
Sequences with .gamma.PNA substitutions based on the above SCD PNAs
can be and have been designed, and include, for example, partial or
complete .gamma.PNA substitution in the Watson-Crick domain,
partial or complete substitutions in the Hoogsteen domain, or a
combination thereof. Exemplary sequences include, but are not
limited to,
TABLE-US-00025 SCD-tcPNA 1A: (SEQ ID NO: 59)
H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-KKK-NH.sub.2 SCD-tcPNA 1B:
(SEQ ID NO: 211) H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-KKK-NH.sub.2
SCD-tcPNA 1C: (SEQ ID NO: 210)
H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAG-KKK-NH.sub.2 SCD-tcPNA 1D:
(SEQ ID NO: 208) H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC-KKK-
NH.sub.2 SCD-tcPNA 1E: (SEQ ID NO: 207)
H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC-KKK- NH.sub.2 SCD-tcPNA
1F: (SEQ ID NO: 206) H-KKK-JJTJTTJ-OOO-CTTCTCCACAGGAGTCAGGTGC-KKK-
NH.sub.2
Underlined residues include a gamma modification, for example,
miniPEG .gamma.PNA substitution. K indicates lysine; J,
pseudoisocytosine (for C) for pH-independent triplex formation.
O,8-amino-2,6,10-trioxaoctanoic acid linkers connecting the
Hoogsteen and Watson-Crick domains of the tcPNAs.
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs.
Publications cited herein and the materials for which they are
cited are specifically incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such
equivalents are intended to be encompassed by the following
claims.
SEQUENCE LISTINGS
1
2161273PRTHomo sapiens 1Met Lys Lys Thr Gln Thr Trp Ile Leu Thr Cys
Ile Tyr Leu Gln Leu1 5 10 15Leu Leu Phe Asn Pro Leu Val Lys Thr Glu
Gly Ile Cys Arg Asn Arg 20 25 30Val Thr Asn Asn Val Lys Asp Val Thr
Lys Leu Val Ala Asn Leu Pro 35 40 45Lys Asp Tyr Met Ile Thr Leu Lys
Tyr Val Pro Gly Met Asp Val Leu 50 55 60Pro Ser His Cys Trp Ile Ser
Glu Met Val Val Gln Leu Ser Asp Ser65 70 75 80Leu Thr Asp Leu Leu
Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95Asn Tyr Ser Ile
Ile Asp Lys Leu Val Asn Ile Val Asp Asp Leu Val 100 105 110Glu Cys
Val Lys Glu Asn Ser Ser Lys Asp Leu Lys Lys Ser Phe Lys 115 120
125Ser Pro Glu Pro Arg Leu Phe Thr Pro Glu Glu Phe Phe Arg Ile Phe
130 135 140Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Val Val Ala Ser
Glu Thr145 150 155 160Ser Asp Cys Val Val Ser Ser Thr Leu Ser Pro
Glu Lys Asp Ser Arg 165 170 175Val Ser Val Thr Lys Pro Phe Met Leu
Pro Pro Val Ala Ala Ser Ser 180 185 190Leu Arg Asn Asp Ser Ser Ser
Ser Asn Arg Lys Ala Lys Asn Pro Pro 195 200 205Gly Asp Ser Ser Leu
His Trp Ala Ala Met Ala Leu Pro Ala Leu Phe 210 215 220Ser Leu Ile
Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Arg225 230 235
240Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu
245 250 255Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu Phe
Gln Glu 260 265 270Val2165PRTHomo sapiens 2Met Glu Gly Ile Cys Arg
Asn Arg Val Thr Asn Asn Val Lys Asp Val1 5 10 15Thr Lys Leu Val Ala
Asn Leu Pro Lys Asp Tyr Met Ile Thr Leu Lys 20 25 30Tyr Val Pro Gly
Met Asp Val Leu Pro Ser His Cys Trp Ile Ser Glu 35 40 45Met Val Val
Gln Leu Ser Asp Ser Leu Thr Asp Leu Leu Asp Lys Phe 50 55 60Ser Asn
Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu65 70 75
80Val Asn Ile Val Asp Asp Leu Val Glu Cys Val Lys Glu Asn Ser Ser
85 90 95Lys Asp Leu Lys Lys Ser Phe Lys Ser Pro Glu Pro Arg Leu Phe
Thr 100 105 110Pro Glu Glu Phe Phe Arg Ile Phe Asn Arg Ser Ile Asp
Ala Phe Lys 115 120 125Asp Phe Val Val Ala Ser Glu Thr Ser Asp Cys
Val Val Ser Ser Thr 130 135 140Leu Ser Pro Glu Lys Asp Ser Arg Val
Ser Val Thr Lys Pro Phe Met145 150 155 160Leu Pro Pro Val Ala
1653273PRTMus musculus 3Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys
Ile Tyr Leu Gln Leu1 5 10 15Leu Leu Phe Asn Pro Leu Val Lys Thr Lys
Glu Ile Cys Gly Asn Pro 20 25 30Val Thr Asp Asn Val Lys Asp Ile Thr
Lys Leu Val Ala Asn Leu Pro 35 40 45Asn Asp Tyr Met Ile Thr Leu Asn
Tyr Val Ala Gly Met Asp Val Leu 50 55 60Pro Ser His Cys Trp Leu Arg
Asp Met Val Ile Gln Leu Ser Leu Ser65 70 75 80Leu Thr Thr Leu Leu
Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95Asn Tyr Ser Ile
Ile Asp Lys Leu Gly Lys Ile Val Asp Asp Leu Val 100 105 110Leu Cys
Met Glu Glu Asn Ala Pro Lys Asn Ile Lys Glu Ser Pro Lys 115 120
125Arg Pro Glu Thr Arg Ser Phe Thr Pro Glu Glu Phe Phe Ser Ile Phe
130 135 140Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Met Val Ala Ser
Asp Thr145 150 155 160Ser Asp Cys Val Leu Ser Ser Thr Leu Gly Pro
Glu Lys Asp Ser Arg 165 170 175Val Ser Val Thr Lys Pro Phe Met Leu
Pro Pro Val Ala Ala Ser Ser 180 185 190Leu Arg Asn Asp Ser Ser Ser
Ser Asn Arg Lys Ala Ala Lys Ala Pro 195 200 205Glu Asp Ser Gly Leu
Gln Trp Thr Ala Met Ala Leu Pro Ala Leu Ile 210 215 220Ser Leu Val
Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Lys225 230 235
240Gln Ser Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu
245 250 255Asp Asn Glu Ile Ser Met Leu Gln Gln Lys Glu Arg Glu Phe
Gln Glu 260 265 270Val4165PRTMus musculus 4Met Lys Glu Ile Cys Gly
Asn Pro Val Thr Asp Asn Val Lys Asp Ile1 5 10 15Thr Lys Leu Val Ala
Asn Leu Pro Asn Asp Tyr Met Ile Thr Leu Asn 20 25 30Tyr Val Ala Gly
Met Asp Val Leu Pro Ser His Cys Trp Leu Arg Asp 35 40 45Met Val Ile
Gln Leu Ser Leu Ser Leu Thr Thr Leu Leu Asp Lys Phe 50 55 60Ser Asn
Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu65 70 75
80Gly Lys Ile Val Asp Asp Leu Val Leu Cys Met Glu Glu Asn Ala Pro
85 90 95Lys Asn Ile Lys Glu Ser Pro Lys Arg Pro Glu Thr Arg Ser Phe
Thr 100 105 110Pro Glu Glu Phe Phe Ser Ile Phe Asn Arg Ser Ile Asp
Ala Phe Lys 115 120 125Asp Phe Met Val Ala Ser Asp Thr Ser Asp Cys
Val Leu Ser Ser Thr 130 135 140Leu Gly Pro Glu Lys Asp Ser Arg Val
Ser Val Thr Lys Pro Phe Met145 150 155 160Leu Pro Pro Val Ala
1655273PRTMus musculus 5Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys
Ile Tyr Leu Gln Leu1 5 10 15Leu Leu Phe Asn Pro Leu Val Lys Thr Gln
Glu Ile Cys Arg Asn Pro 20 25 30Val Thr Asp Asn Val Lys Asp Ile Thr
Lys Leu Val Ala Asn Leu Pro 35 40 45Asn Asp Tyr Met Ile Thr Leu Asn
Tyr Val Ala Gly Met Asp Val Leu 50 55 60Pro Ser His Cys Trp Leu Arg
Asp Met Val Thr His Leu Ser Val Ser65 70 75 80Leu Thr Thr Leu Leu
Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 85 90 95Asn Tyr Ser Ile
Ile Asp Lys Leu Gly Lys Ile Val Asp Asp Leu Val 100 105 110Ala Cys
Met Glu Glu Asn Ala Pro Lys Asn Val Lys Glu Ser Leu Lys 115 120
125Lys Pro Glu Thr Arg Asn Phe Thr Pro Glu Glu Phe Phe Ser Ile Phe
130 135 140Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Met Val Ala Ser
Asp Thr145 150 155 160Ser Asp Cys Val Leu Ser Ser Thr Leu Gly Pro
Glu Lys Asp Ser Arg 165 170 175Val Ser Val Thr Lys Pro Phe Met Leu
Pro Pro Val Ala Ala Ser Ser 180 185 190Leu Arg Asn Asp Ser Ser Ser
Ser Asn Arg Lys Ala Ala Lys Ser Pro 195 200 205Glu Asp Pro Gly Leu
Gln Trp Thr Ala Met Ala Leu Pro Ala Leu Ile 210 215 220Ser Leu Val
Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Lys225 230 235
240Gln Ser Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu
245 250 255Asp Asn Glu Ile Ser Met Leu Gln Gln Lys Glu Arg Glu Phe
Gln Glu 260 265 270Val6165PRTRattus rattus 6Met Gln Glu Ile Cys Arg
Asn Pro Val Thr Asp Asn Val Lys Asp Ile1 5 10 15Thr Lys Leu Val Ala
Asn Leu Pro Asn Asp Tyr Met Ile Thr Leu Asn 20 25 30Tyr Val Ala Gly
Met Asp Val Leu Pro Ser His Cys Trp Leu Arg Asp 35 40 45Met Val Thr
His Leu Ser Val Ser Leu Thr Thr Leu Leu Asp Lys Phe 50 55 60Ser Asn
Ile Ser Glu Gly Leu Ser Asn Tyr Ser Ile Ile Asp Lys Leu65 70 75
80Gly Lys Ile Val Asp Asp Leu Val Ala Cys Met Glu Glu Asn Ala Pro
85 90 95Lys Asn Val Lys Glu Ser Leu Lys Lys Pro Glu Thr Arg Asn Phe
Thr 100 105 110Pro Glu Glu Phe Phe Ser Ile Phe Asn Arg Ser Ile Asp
Ala Phe Lys 115 120 125Asp Phe Met Val Ala Ser Asp Thr Ser Asp Cys
Val Leu Ser Ser Thr 130 135 140Leu Gly Pro Glu Lys Asp Ser Arg Val
Ser Val Thr Lys Pro Phe Met145 150 155 160Leu Pro Pro Val Ala
165711PRTArtificial SequenceSynthetic peptide 7Tyr Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg1 5 1089PRTArtificial SequenceSynthetic
Peptide 8Arg Lys Lys Arg Arg Gln Arg Arg Arg1 5916PRTArtificial
SequenceSynthetic Peptide 9Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg
Arg Met Lys Trp Lys Lys1 5 10 151019PRTArtificial SequenceSynthetic
Peptide 10His His His His Arg Lys Lys Arg Arg Gln Arg Arg Arg Arg
His His1 5 10 15His His His1130PRTArtificial SequenceSynthetic
Peptide 11Met Val Lys Ser Lys Ile Gly Ser Trp Ile Leu Val Leu Phe
Val Ala1 5 10 15Met Trp Ser Asp Val Gly Leu Cys Lys Lys Arg Pro Lys
Pro 20 25 301227PRTArtificial SequenceSynthetic Peptide 12Gly Ala
Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly1 5 10 15Ala
Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25136060DNAHomo sapiens
13aaagctcttg ctttgacaat tttggtcttt cagaatacta taaatataac ctatattata
60atttcataaa gtctgtgcat tttctttgac ccaggatatt tgcaaaagac atattcaaac
120ttccgcagaa cactttattt cacatataca tgcctcttat atcagggatg
tgaaacaggg 180tcttgaaaac tgtctaaatc taaaacaatg ctaatgcagg
tttaaattta ataaaataaa 240atccaaaatc taacagccaa gtcaaatctg
tatgttttaa catttaaaat attttaaaga 300cgtcttttcc caggattcaa
catgtgaaat cttttctcag ggatacacgt gtgcctagat 360cctcattgct
ttagtttttt acagaggaat gaatataaaa agaaaatact taaattttat
420ccctcttacc tctataatca tacataggca taatttttta acctaggctc
cagatagcca 480tagaagaacc aaacactttc tgcgtgtgtg agaataatca
gagtgagatt ttttcacaag 540tacctgatga gggttgagac aggtagaaaa
agtgagagat ctctatttat ttagcaataa 600tagagaaagc atttaagaga
ataaagcaat ggaaataaga aatttgtaaa tttccttctg 660ataactagaa
atagaggatc cagtttcttt tggttaacct aaattttatt tcattttatt
720gttttatttt attttatttt attttatttt gtgtaatcgt agtttcagag
tgttagagct 780gaaaggaaga agtaggagaa acatgcaaag taaaagtata
acactttcct tactaaaccg 840actgggtttc caggtagggg caggattcag
gatgactgac agggccctta gggaacactg 900agaccctacg ctgacctcat
aaatgcttgc tacctttgct gttttaatta catcttttaa 960tagcaggaag
cagaactctg cacttcaaaa gtttttcctc acctgaggag ttaatttagt
1020acaaggggaa aaagtacagg gggatgggag aaaggcgatc acgttgggaa
gctatagaga 1080aagaagagta aattttagta aaggaggttt aaacaaacaa
aatataaaga gaaataggaa 1140cttgaatcaa ggaaatgatt ttaaaacgca
gtattcttag tggactagag gaaaaaaata 1200atctgagcca agtagaagac
cttttcccct cctaccccta ctttctaagt cacagaggct 1260ttttgttccc
ccagacactc ttgcagatta gtccaggcag aaacagttag atgtccccag
1320ttaacctcct atttgacacc actgattacc ccattgatag tcacactttg
ggttgtaagt 1380gactttttat ttatttgtat ttttgactgc attaagaggt
ctctagtttt ttatctcttg 1440tttcccaaaa cctaataagt aactaatgca
cagagcacat tgatttgtat ttattctatt 1500tttagacata atttattagc
atgcatgagc aaattaagaa aaacaacaac aaatgaatgc 1560atatatatgt
atatgtatgt gtgtatatat acacatatat atatatattt tttttctttt
1620cttaccagaa ggttttaatc caaataagga gaagatatgc ttagaactga
ggtagagttt 1680tcatccattc tgtcctgtaa gtattttgca tattctggag
acgcaggaag agatccatct 1740acatatccca aagctgaatt atggtagaca
aagctcttcc acttttagtg catcaatttc 1800ttatttgtgt aataagaaaa
ttgggaaaac gatcttcaat atgcttacca agctgtgatt 1860ccaaatatta
cgtaaataca cttgcaaagg aggatgtttt tagtagcaat ttgtactgat
1920ggtatggggc caagagatat atcttagagg gagggctgag ggtttgaagt
ccaactccta 1980agccagtgcc agaagagcca aggacaggta cggctgtcat
cacttagacc tcaccctgtg 2040gagccacacc ctagggttgg ccaatctact
cccaggagca gggagggcag gagccagggc 2100tgggcataaa agtcagggca
gagccatcta ttgcttacat ttgcttctga cacaactgtg 2160ttcactagca
acctcaaaca gacaccatgg tgcacctgac tcctgaggag aagtctgccg
2220ttactgccct gtggggcaag gtgaacgtgg atgaagttgg tggtgaggcc
ctgggcaggt 2280tggtatcaag gttacaagac aggtttaagg agaccaatag
aaactgggca tgtggagaca 2340gagaagactc ttgggtttct gataggcact
gactctctct gcctattggt ctattttccc 2400acccttaggc tgctggtggt
ctacccttgg acccagaggt tctttgagtc ctttggggat 2460ctgtccactc
ctgatgctgt tatgggcaac cctaaggtga aggctcatgg caagaaagtg
2520ctcggtgcct ttagtgatgg cctggctcac ctggacaacc tcaagggcac
ctttgccaca 2580ctgagtgagc tgcactgtga caagctgcac gtggatcctg
agaacttcag ggtgagtcta 2640tgggaccctt gatgttttct ttccccttct
tttctatggt taagttcatg tcataggaag 2700gggagaagta acagggtaca
gtttagaatg ggaaacagac gaatgattgc atcagtgtgg 2760aagtctcagg
atcgttttag tttcttttat ttgctgttca taacaattgt tttcttttgt
2820ttaattcttg ctttcttttt ttttcttctc cgcaattttt actattatac
ttaatgcctt 2880aacattgtgt ataacaaaag gaaatatctc tgagatacat
taagtaactt aaaaaaaaac 2940tttacacagt ctgcctagta cattactatt
tggaatatat gtgtgcttat ttgcatattc 3000ataatctccc tactttattt
tcttttattt ttaattgata cataatcatt atacatattt 3060atgggttaaa
gtgtaatgtt ttaatatgtg tacacatatt gaccaaatca gggtaatttt
3120gcatttgtaa ttttaaaaaa tgctttcttc ttttaatata cttttttgtt
tatcttattt 3180ctaatacttt ccctaatctc tttctttcag ggcaataatg
atacaatgta tcatgcctct 3240ttgcaccatt ctaaagaata acagtgataa
tttctgggtt aaggcaatag caatatttct 3300gcatataaat atttctgcat
ataaattgta actgatgtaa gaggtttcat attgctaata 3360gcagctacaa
tccagctacc attctgcttt tattttatgg ttgggataag gctggattat
3420tctgagtcca agctaggccc ttttgctaat catgttcata cctcttatct
tcctcccaca 3480gctcctgggc aacgtgctgg tctgtgtgct ggcccatcac
tttggcaaag aattcacccc 3540accagtgcag gctgcctatc agaaagtggt
ggctggtgtg gctaatgccc tggcccacaa 3600gtatcactaa gctcgctttc
ttgctgtcca atttctatta aaggttcctt tgttccctaa 3660gtccaactac
taaactgggg gatattatga agggccttga gcatctggat tctgcctaat
3720aaaaaacatt tattttcatt gcaatgatgt atttaaatta tttctgaata
ttttactaaa 3780aagggaatgt gggaggtcag tgcatttaaa acataaagaa
atgaagagct agttcaaacc 3840ttgggaaaat acactatatc ttaaactcca
tgaaagaagg tgaggctgca aacagctaat 3900gcacattggc aacagccctg
atgcctatgc cttattcatc cctcagaaaa ggattcaagt 3960agaggcttga
tttggaggtt aaagttttgc tatgctgtat tttacattac ttattgtttt
4020agctgtcctc atgaatgtct tttcactacc catttgctta tcctgcatct
ctcagccttg 4080actccactca gttctcttgc ttagagatac cacctttccc
ctgaagtgtt ccttccatgt 4140tttacggcga gatggtttct cctcgcctgg
ccactcagcc ttagttgtct ctgttgtctt 4200atagaggtct acttgaagaa
ggaaaaacag ggggcatggt ttgactgtcc tgtgagccct 4260tcttccctgc
ctcccccact cacagtgacc cggaatctgc agtgctagtc tcccggaact
4320atcactcttt cacagtctgc tttggaagga ctgggcttag tatgaaaagt
taggactgag 4380aagaatttga aagggggctt tttgtagctt gatattcact
actgtcttat taccctatca 4440taggcccacc ccaaatggaa gtcccattct
tcctcaggat gtttaagatt agcattcagg 4500aagagatcag aggtctgctg
gctcccttat catgtccctt atggtgcttc tggctctgca 4560gttattagca
tagtgttacc atcaaccacc ttaacttcat ttttcttatt caatacctag
4620gtaggtagat gctagattct ggaaataaaa tatgagtctc aagtggtcct
tgtcctctct 4680cccagtcaaa ttctgaatct agttggcaag attctgaaat
caaggcatat aatcagtaat 4740aagtgatgat agaagggtat atagaagaat
tttattatat gagagggtga aacctaaaat 4800gaaatgaaat cagacccttg
tcttacacca taaacaaaaa taaatttgaa tgggttaaag 4860aattaaacta
agacctaaaa ccataaaaat ttttaaagaa atcaaaagaa gaaaattcta
4920atattcatgt tgcagccgtt ttttgaattt gatatgagaa gcaaaggcaa
caaaaggaaa 4980aataaagaag tgaggctaca tcaaactaaa aaatttccac
acaaaaaaga aaacaatgaa 5040caaatgaaag gtgaaccatg aaatggcata
tttgcaaacc aaatatttct taaatatttt 5100ggttaatatc caaaatatat
aagaaacaca gatgattcaa taacaaacaa aaaattaaaa 5160ataggaaaat
aaaaaaatta aaaagaagaa aatcctgcca tttatgcgag aattgatgaa
5220cctggaggat gtaaaactaa gaaaaataag cctgacacaa aaagacaaat
actacacaac 5280cttgctcata tgtgaaacat aaaaaagtca ctctcatgga
aacagacagt agaggtatgg 5340tttccagggg ttgggggtgg gagaatcagg
aaactattac tcaaagggta taaaatttca 5400gttatgtggg atgaataaat
tctagatatc taatgtacag catcgtgact gtagttaatt 5460gtactgtaag
tatatttaaa atttgcaaag agagtagatt tttttgtttt tttagatgga
5520gttttgctct tgttgtccag gctggagtgc aatggcaaga tcttggctca
ctgcaacctc 5580cgcctcctgg gttcaagcaa atctcctgcc tcagcctccc
gagtagctgg gattacaggc 5640atgcgacacc atgcccagct aattttgtat
ttttagtaga gacggggttt ctccatgttg 5700gtcaggctga tccgcctcct
cggccaccaa agggctggga ttacaggcgt gaccaccggg 5760cctggccgag
agtagatctt aaaagcattt accacaagaa aaaggtaact atgtgagata
5820atgggtatgt taattagctt gattgtggta atcatttcac aaggtataca
tatattaaaa 5880catcatgttg tacaccttaa atatatacaa tttttatttg
tgaatgatac ctcaataaag 5940ttgaagaata ataaaaaaga atagacatca
catgaattaa aaaactaaaa aataaaaaaa 6000tgcatcttga tgattagaat
tgcattcttg atttttcaga
tacaaatatc catttgactg 606014850DNAHomo sapiens 14gtgagtctat
gggacccttg atgttttctt tccccttctt ttctatggtt aagttcatgt 60cataggaagg
ggagaagtaa cagggtacag tttagaatgg gaaacagacg aatgattgca
120tcagtgtgga agtctcagga tcgttttagt ttcttttatt tgctgttcat
aacaattgtt 180ttcttttgtt taattcttgc tttctttttt tttcttctcc
gcaattttta ctattatact 240taatgcctta acattgtgta taacaaaagg
aaatatctct gagatacatt aagtaactta 300aaaaaaaact ttacacagtc
tgcctagtac attactattt ggaatatatg tgtgcttatt 360tgcatattca
taatctccct actttatttt cttttatttt taattgatac ataatcatta
420tacatattta tgggttaaag tgtaatgttt taatatgtgt acacatattg
accaaatcag 480ggtaattttg catttgtaat tttaaaaaat gctttcttct
tttaatatac ttttttgttt 540atcttatttc taatactttc cctaatctct
ttctttcagg gcaataatga tacaatgtat 600catgcctctt tgcaccattc
taaagaataa cagtgataat ttctgggtta aggcaatagc 660aatatttctg
catataaata tttctgcata taaattgtaa ctgatgtaag aggtttcata
720ttgctaatag cagctacaat ccagctacca ttctgctttt attttatggt
tgggataagg 780ctggattatt ctgagtccaa gctaggccct tttgctaatc
atgttcatac ctcttatctt 840cctcccacag 8501512DNAArtificial
SequenceSynthetic Primer 15gaaagaaaga ga 121618DNAArtificial
SequenceSynthetic Primer 16tgccctgaaa gaaagaga 18177DNAArtificial
SequenceSynthetic Primer 17ggagaaa 71817DNAArtificial
SequenceSynthetic Primer 18agaatggtgc aaagagg 17197DNAArtificial
SequenceSynthetic Primer 19aaaaggg 72018DNAArtificial
SequenceSynthetic Primer 20acatgattag caaaaggg 182112DNAArtificial
SequenceSynthetic Primer 21ctttctttct ct 122212DNAArtificial
SequenceSynthetic Primer 22tctctttctt tc 122318DNAArtificial
SequenceSynthetic Primer 23tctctttctt tcagggca 18246DNAArtificial
SequenceSynthetic Primer 24tttccc 6257DNAArtificial
SequenceSynthetic Primer 25ccctttt 72618DNAArtificial
SequenceSynthetic Primer 26cccttttgct aatcatgt 18277DNAArtificial
SequenceSynthetic Primer 27tttctcc 7287DNAArtificial
SequenceSynthetic Primer 28cctcttt 72917DNAArtificial
SequenceSynthetic Primer 29cctctttgca ccattct 173012DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(5)..(5)n =
Pseudoisocytosinemisc_feature(9)..(9)n =
Pseudoisocytosinemisc_feature(11)..(11)n = Pseudoisocytosine
30ntttntttnt nt 12317DNAArtificial SequenceSynthetic
Primermisc_feature(5)..(7)n = Pseudoisocytosine 31ttttnnn
7327DNAArtificial SequenceSynthetic Primermisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(6)..(7)n = Pseudoisocytosine
32tttntnn 73330DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(5)..(5)n =
Pseudoisocytosinemisc_feature(9)..(9)n =
Pseudoisocytosinemisc_feature(11)..(11)n =
Pseudoisocytosinemisc_feature(12)..(13)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(30)..(30)Linked
to lys-lys-lys 33ntttntttnt nttctctttc tttcagggca
303425DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(5)..(7)n
= Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 34ttttnnnccc ttttgctaat catgt 253524DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(6)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(24)..(24)Linked
to lys-lys-lys 35tttntnncct ctttgcacca ttct 243610DNAArtificial
SequenceSynthetic Primermisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(10)..(10)n = Pseudoisocytosine
36tnttttnttn 103710DNAArtificial SequenceSynthetic Primer
37cttcttttct 103810DNAArtificial SequenceSynthetic
Primermisc_feature(3)..(3)n =
Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(10)..(10)n = Pseudoisocytosine
38ttnttntttn 103910DNAArtificial SequenceSynthetic Primer
39ctttcttctt 104010DNAArtificial SequenceSynthetic
Primermisc_feature(1)..(3)n =
Pseudoisocytosinemisc_feature(5)..(6)n =
Pseudoisocytosinemisc_feature(9)..(9)n = Pseudoisocytosine
40nnntnnttnt 104110DNAArtificial SequenceSynthetic Primer
41tcttcctccc 104220DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(2)..(2)n
= Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(10)..(10)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(20)..(20)Linked
to lys-lys-lys 42tnttttnttn cttcttttct 204320DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(3)..(3)n =
Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(10)..(10)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(20)..(20)Linked
to lys-lys-lys 43ttnttntttn ctttcttctt 204420DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(3)n =
Pseudoisocytosinemisc_feature(5)..(6)n =
Pseudoisocytosinemisc_feature(9)..(9)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(20)..(20)Linked
to lys-lys-lys 44nnntnnttnt tcttcctccc 20457DNAArtificial
SequenceSynthetic Primer 45cctcttc 7467DNAArtificial
SequenceSynthetic Primer 46cttctcc 74715DNAArtificial
SequenceSynthetic Primer 47cttctccaaa ggagt 154818DNAArtificial
SequenceSynthetic Primer 48cttctccaca ggagtcag 18497DNAArtificial
SequenceSynthetic Primer 49ttcctct 7507DNAArtificial
SequenceSynthetic Primer 50tctcctt 75115DNAArtificial
SequenceSynthetic Primer 51tctccttaaa cctgt 15528DNAArtificial
SequenceSynthetic Primer 52tctcttct 8538DNAArtificial
SequenceSynthetic Primer 53tcttctct 85416DNAArtificial
SequenceSynthetic Primer 54tcttctctgt ctccac 165518DNAArtificial
SequenceSynthetic Primer 55tcttctctgt ctccacat 18567DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n = Pseudoisocytosine
56nntnttn 75722DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(3)..(4)n
= Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(22)..(22)Linked
to lys-lys-lys 57ttnntnttct ccttaaacct gt 225824DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(8)..(9)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(24)..(24)Linked
to lys-lys-lys 58tntnttnttc ttctctgtct ccac 245925DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 59nntnttnctt ctccacagga gtcag 256026DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(8)..(9)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(26)..(26)Linked
to lys-lys-lys 60tntnttnttc ttctctgtct ccacat 266151DNAArtificial
SequenceSynthetic Primer 61gttcagcgtg tccggcgagg gcgaggtgag
tctatgggac ccttgatgtt t 516251DNAArtificial SequenceSynthetic
Primer 62aaacatcaag ggtcccatag actcacctcg ccctcgccgg acacgctgaa c
516370DNAArtificial SequenceSynthetic Primer 63cttgccccac
agggcagtaa cggcagattt ttcttccggc gttaaatgca ccatggtgtc 60tgtttgaggt
706451DNAArtificial SequenceSynthetic Primer 64acagacacca
tggtgcacct gactcctgag gagaagtctg ccgttactgc c 516560DNAArtificial
SequenceSynthetic Primer 65aaagaataac agtgataatt tctgggttaa
ggcaatagca atatctctgc atataaatat 606622DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(22)..(22)Linked
to lys-lys-lys 66nntnttnctt ctccaaagga gt 226722DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(3)..(4)n =
Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(22)..(22)Linked
to lys-lys-lys 67ttnntnttct ccttaaacct gt 226824DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(8)..(9)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculessmisc_feature(24)..(4)Linked
to lys-lys-lys 68tntnttnttc ttctctgtct ccac 246925DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(5)n =
Pseudoisocytosinemisc_feature(8)..(9)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 69tntnnttttt tcctctatgg gtaag 25708DNAArtificial
SequenceSynthetic Primer 70tttcctct 87117DNAArtificial
SequenceSynthetic Primer 71tttcctctat gggtaag 17728DNAArtificial
SequenceSynthetic Primer 72agaggaaa 87317DNAArtificial
SequenceSynthetic Primer 73cttacccata gaggaaa 17748DNAArtificial
SequenceSynthetic Primer 74agaagagg 87517DNAArtificial
SequenceSynthetic Primer 75atgccaacta gaagagg 17768DNAArtificial
SequenceSynthetic Primer 76cctcttct 87717DNAArtificial
SequenceSynthetic Primer 77cctcttctag ttggcat 17789DNAArtificial
SequenceSynthetic Primer 78ctttccctt 97918DNAArtificial
SequenceSynthetic Primer 79ctttcccttg tatctttt 18809DNAArtificial
SequenceSynthetic Primer 80aagggaaag 98118DNAArtificial
SequenceSynthetic Primer 81aaaagataca agggaaag 18828DNAArtificial
SequenceSynthetic Primer 82tctccttt 8838DNAArtificial
SequenceSynthetic Primer 83tttcctct 88417DNAArtificial
SequenceSynthetic Primer 84tttcctctat gggtaag 17858DNAArtificial
SequenceSynthetic Primer 85tcttctcc 8868DNAArtificial
SequenceSynthetic Primer 86cctcttct 88717DNAArtificial
SequenceSynthetic Primer 87cctcttctag ttggcat 17889DNAArtificial
SequenceSynthetic Primer 88ttccctttc 9899DNAArtificial
SequenceSynthetic Primer 89ctttccctt 99018DNAArtificial
SequenceSynthetic Primer 90ctttcccttg tatctttt 18918DNAArtificial
SequenceSynthetic Primermisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(5)n = Pseudoisocytosine
91tntnnttt 8929DNAArtificial SequenceSynthetic
Primermisc_feature(3)..(5)n =
Pseudoisocytosinemisc_feature(9)..(9)n = Pseudoisocytosine
92ttnnntttn 99325DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(2)..(2)n
= Pseudoisocytosinemisc_feature(4)..(5)n =
Pseudoisocytosinemisc_feature(8)..(9)Linked by three 8-amino-2,
6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 93tntnnttttt tcctctatgg gtaag 259425DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(5)..(5)n =
Pseudoisocytosinemisc_feature(7)..(8)n =
Pseudoisocytosinemisc_feature(8)..(9)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 94tnttntnncc tcttctagtt ggcat 259527DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(3)..(5)n =
Pseudoisocytosinemisc_feature(9)..(9)n =
Pseudoisocytosinemisc_feature(9)..(10)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(27)..(27)Linked
to lys-lys-lys 95ttnnntttnc tttcccttgt atctttt 279661DNAArtificial
SequenceSynthetic Primer 96ttctgtatct atattcatca taggaaacac
caaagataat gttctcctta atggtgccag 60g 619710DNAArtificial
SequenceSynthetic Primer 97cttcctcttt 109810DNAArtificial
SequenceSynthetic Primer 98tttctccttc 109918DNAArtificial
SequenceSynthetic Primer 99tttctccttc agtgttca 181007DNAArtificial
SequenceSynthetic Primer 100ttttcct 71017DNAArtificial
SequenceSynthetic Primer 101tcctttt 710220DNAArtificial
SequenceSynthetic Primer 102tccttttgct cacctgtggt
2010310DNAArtificial SequenceSynthetic Primer 103tcttttttcc
1010410DNAArtificial SequenceSynthetic Primer 104ccttttttct
1010518DNAArtificial SequenceSynthetic Primer 105ccttttttct
ggctaagt 1810610DNAArtificial SequenceSynthetic
Primermisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(4)..(5)n =
Pseudoisocytosinemisc_feature(7)..(7)n = Pseudoisocytosine
106nttnntnttt 101077DNAArtificial SequenceSynthetic
Primermisc_feature(5)..(6)n = Pseudoisocytosine 107ttttnnt
710810DNAArtificial SequenceSynthetic Primermisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(9)..(10)n = Pseudoisocytosine
108tnttttttnn 1010958DNAArtificial SequenceSynthetic
Primermisc_feature(1)..(2)Optional phosphorothioate internucleoside
linkagemisc_feature(2)..(3)Optional phosphorothioate
internucleoside linkagemisc_feature(3)..(4)Optional
phosphorothioate internucleoside
linkagemisc_feature(55)..(56)Optional phosphorothioate
internucleoside linkagemisc_feature(56)..(57)Optional
phosphorothioate internucleoside
linkagemisc_feature(57)..(58)Optional phosphorothioate
internucleoside linkage 109tgggattcaa taaccttgca gacagtggag
gaaggccttt ggcgtgatac cacaggtg 581107DNAArtificial
SequenceSynthetic Primer 110tcttttt 71117DNAArtificial
SequenceSynthetic Primer 111tttttct 711218DNAArtificial
SequenceSynthetic Primer 112tttttctgta atttttaa 181139DNAArtificial
SequenceSynthetic Primer 113tctctttct 91149DNAArtificial
SequenceSynthetic Primer 114tctttctct 911517DNAArtificial
SequenceSynthetic Primer 115tctttctctg caaactt 171167DNAArtificial
SequenceSynthetic Primer 116tttcttt 711718DNAArtificial
SequenceSynthetic Primer 117tttctttaag aacgagca 181187DNAArtificial
SequenceSynthetic Primermisc_feature(2)..(2)n = Pseudoisocytosine
118tnttttt 71199DNAArtificial SequenceSynthetic
Primermisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(8)..(8)n = Pseudoisocytosine
119tntntttnt 91207DNAArtificial SequenceSynthetic
Primermisc_feature(4)..(4)n = Pseudoisocytosine 120tttnttt
712125DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(2)..(2)n
= Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 121tntttttttt ttctgtaatt tttaa 2512226DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(8)..(8)n =
Pseudoisocytosinemisc_feature(9)..(10)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(26)..(26)Linked
to lys-lys-lys 122tntntttntt ctttctctgc aaactt 2612325DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 123tttntttttt ctttaagaac gagca 2512460DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(2)Optional
phosphorothioate internucleoside
linkagemisc_feature(2)..(3)Optional phosphorothioate
internucleoside linkagemisc_feature(3)..(4)Optional
phosphorothioate internucleoside
linkagemisc_feature(57)..(58)Optional phosphorothioate
internucleoside linkagemisc_feature(58)..(59)Optional
phosphorothioate internucleoside
linkagemisc_feature(59)..(60)Optional phosphorothioate
internucleoside linkage 124aagtttgcag agaaagataa tatagtcctt
ggagaaggag gaatcaccct gagtggaggt 6012510DNAArtificial
SequenceSynthetic Primer 125ctcttcttct 1012610DNAArtificial
SequenceSynthetic Primer 126tcttcttctc 1012715DNAArtificial
SequenceSynthetic Primer 127tcttcttctc atttc 151285DNAArtificial
SequenceSynthetic Primer 128cttct 51295DNAArtificial
SequenceSynthetic Primer 129tcttc 513010DNAArtificial
SequenceSynthetic Primer 130tcttcttctc 1013115DNAArtificial
SequenceSynthetic Primer 131tcttcttctc atttc 1513210DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(3)..(3)n =
Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(9)..(9)n = Pseudoisocytosine
132ntnttnttnt 101335DNAArtificial SequenceSynthetic
Primermisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(4)..(4)n = Pseudoisocytosine 133nttnt
513425DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(1)n
= Pseudoisocytosinemisc_feature(3)..(3)n =
Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(9)..(9)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 134ntnttnttnt tcttcttctc atttc 2513520DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(5)..(6)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(20)..(20)Linked
to lys-lys-lys 135nttnttcttc ttctcatttc 2013660DNAArtificial
SequenceSynthetic Primer 136attcccgagt agcagatgac catgacagct
tagggcagga ccagccccaa gatgactatc 6013760DNAArtificial
SequenceSynthetic Primer 137tttaggattc ccgagtagca gatgacccct
cagagcagcg gcaggaccag ccccaagatg 6013865DNAArtificial
SequenceSynthetic Primer 138gatgactatc tttaatgtct ggaaattctt
ccagaattaa ttaagactgt atggaaaatg 60agagc 6513966DNAArtificial
SequenceSynthetic Primer 139ccccaagatg actatcttta atgtctggaa
cgatcatcag aattgatact gactgtatgg 60aaaatg 6614065DNAArtificial
SequenceSynthetic Primer 140gatgactatc tttaatgtct ggaaattcta
ctagaattga tactgactgt atggaaaatg 60agagc 6514112DNAArtificial
SequenceSynthetic Primer 141ctgctcggaa ga 1214212DNAArtificial
SequenceSynthetic Primer 142tcttccgagc ag 1214315DNAArtificial
SequenceSynthetic Primer 143ccttcaccaa gggga 1514415DNAArtificial
SequenceSynthetic Primer 144tccccttggt gaagg 151457DNAArtificial
SequenceSynthetic Primer 145ttcccct 71467DNAArtificial
SequenceSynthetic Primer 146tcccctt 714715DNAArtificial
SequenceSynthetic Primer 147tccccttggt gaagg 151487DNAArtificial
SequenceSynthetic Primermisc_feature(3)..(6)n = Pseudoisocytosine
148ttnnnnt 714963DNAArtificial SequenceSynthetic PRimer
149aggacggtcc cggcctgcga cacttccgcc cataattgtt cttcatctgc
ggggcggggg 60ggg 631506DNAArtificial SequenceSynthetic Primer
150ccttct 61516DNAArtificial SequenceSynthetic Primer 151tcttcc
615212DNAArtificial SequenceSynthetic Primer 152tcttccgagc ag
1215318DNAArtificial SequenceSynthetic
Primermisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(2)n =
Pseudoisocytosinemisc_feature(5)..(5)n =
Pseudoisocytosinemisc_feature(6)..(7)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(18)..(18)Linked
to lys-lys-lys 153nnttnttctt ccgagcag 1815467DNAArtificial
SequenceSynthetic Primer 154gggacggcgc ccacataggc caaattcaat
tgctgatccc agcttaagac gtactggtca 60gcctggc 6715528DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(4)..(5)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(28)..(28)Linked
to lys-lys-lys 155nttnntnttt tttctccttc agtgttca
2815627DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(5)..(6)n
= Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(27)..(27)Linked
to lys-lys-lys 156ttttnnttcc ttttgctcac ctgtggt
2715728DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(2)..(2)n
= Pseudoisocytosinemisc_feature(9)..(10)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(28)..(28)Linked
to lys-lys-lys 157tnttttttnn ccttttttct ggctaagt
2815830DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(3)..(3)n
= Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(10)..(10)n =
Pseudoisocytosinemisc_feature(12)..(12)n =
Pseudoisocytosinemisc_feature(12)..(13)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(30)..(30)Linked
to lys-lys-lys 158ttntttnttn tnctcttctt tcttgacagg
3015922DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(3)..(6)n
= Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(22)..(22)Linked
to lys-lys-lys 159ttnnnnttcc ccttggtgaa gg 2216022DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(22)..(22)Linked
to lys-lys-lys 160nntnttnctt ctccaaagga gt 2216165DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(2)phosphorothioate
internucleoside linkagemisc_feature(2)..(3)phosphorothioate
internucleoside linkagemisc_feature(3)..(4)phosphorothioate
internucleoside linkagemisc_feature(62)..(63)phosphorothioate
internucleoside linkagemisc_feature(63)..(64)phosphorothioate
internucleoside linkagemisc_feature(64)..(65)phosphorothioate
internucleoside linkage 161ttgccccaca gggcagtaac ggcagacttc
tcctcaggag tcaggtgcac catggtgtct 60gtttg 6516230DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(1)n =
Pseudoisocytosinemisc_feature(5)..(5)n =
Pseudoisocytosinemisc_feature(9)..(9)n =
Pseudoisocytosinemisc_feature(11)..(11)n =
Pseudoisocytosinemisc_feature(12)..(13)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(30)..(30)Linked
to lys-lys-lys 162ntttntttnt nttctctttc tttcagggca
3016310DNAArtificial SequenceSynthetic Primer 163gaaggagaaa
101647DNAArtificial SequenceSynthetic Primer 164aaaagga
71659DNAArtificial SequenceSynthetic Primer 165agaaaaaag
91667DNAArtificial SequenceSynthetic Primer 166agaaaaa
71679DNAArtificial SequenceSynthetic Primer 167agagaaaga
91687DNAArtificial SequenceSynthetic Primer 168aaagaaa
716928DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(1)n
= Pseudoisocytosinemisc_feature(4)..(5)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(28)..(28)Linked
to lys-lys-lys 169nttnntnttt tttctccttc agtgttca
2817027DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(5)..(6)n
= Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(27)..(27)Linked
to lys-lys-lys 170ttttnnttcc ttttgctcac ctgtggt
2717128DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(2)..(2)n
= Pseudoisocytosinemisc_feature(9)..(10)n =
Pseudoisocytosinemisc_feature(10)..(11)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(28)..(28)Linked
to lys-lys-lys 171tnttttttnn ccttttttct ggctaagt
2817225DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(2)..(2)n
= Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2,
6, 10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 172tntttttttt ttctgtaatt tttaa 2517326DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(8)..(8)n =
Pseudoisocytosinemisc_feature(9)..(10)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(26)..(26)Linked
to lys-lys-lys 173tntntttntt ctttctctgc aaactt 2617425DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 174tttntttttt ctttaagaac gagca 2517560DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(2)Optional
phosphorothioate internucleoside
linkagemisc_feature(2)..(3)Optional phosphorothioate
internucleoside linkagemisc_feature(3)..(4)Optional
phosphorothioate internucleoside
linkagemisc_feature(57)..(58)Optional phosphorothioate
internucleoside linkagemisc_feature(58)..(59)Optional
phosphorothioate internucleoside
linkagemisc_feature(59)..(60)Optional phosphorothioate
internucleoside linkage 175aaagaataac agtgataatt tctgggttaa
ggcaatagca atatctctgc atataaatat 6017620DNAArtificial
SequenceSynthetic Primer 176ttgggaaatt tttaaggcga
2017725DNAArtificial SequenceSynthetic Primer 177tgatgacatc
aagaaggtgg tgaag 2517823DNAArtificial SequenceSynthetic Primer
178tccttggagg ccatgtgggc cat 2317920DNAArtificial SequenceSynthetic
Primer 179tatcatgcct ctttgcacca 2018024DNAArtificial
SequenceSynthetic Primer 180agcaatatga aacctcttac atca
2418123DNAArtificial SequenceSynthetic Primer 181agataattat
tgcctcccac tgc 2318220DNAArtificial SequenceSynthetic Primer
182aatggaaggg catgcagtca 2018320DNAArtificial SequenceSynthetic
Primer 183cccaatcctg aatcctggct 2018422DNAArtificial
SequenceSynthetic Primer 184catactgatg tctgtggctt ga
2218521DNAArtificial SequenceSynthetic Primer 185aagctcaaac
ctaccagacc a 2118621DNAArtificial SequenceSynthetic Primer
186agctggaagc ttcttcagtc a 2118720DNAArtificial SequenceSynthetic
Primer 187ccctctgtgg actgaggaag 2018821DNAArtificial
SequenceSynthetic Primer 188tgatgagcta cgggtatgtg a
2118923DNAArtificial SequenceSynthetic Primer 189caaaaagcct
taagcaaaca ctc 2319022DNAArtificial SequenceSynthetic Primer
190tctctccctc agcatctatt cc 2219125DNAArtificial SequenceSynthetic
Primer 191tgtgtttgtt tatggatact tgagc 2519220DNAArtificial
SequenceSynthetic Primer 192gcatgcacaa taaaggcact
2019322DNAArtificial SequenceSynthetic Primer 193catgggaaac
agtcaaaaga aa 2219420DNAArtificial SequenceSynthetic Primer
194tgtaggtttc cccacagctt 2019518DNAArtificial SequenceSynthetic
Primer 195tgccctgaaa gaaagaga 1819618DNAArtificial
SequenceSynthetic Primer 196agccctgaaa gaaagaga
1819718DNAArtificial SequenceSynthetic Primer 197gaacctgaaa
gaaagaga 1819818DNAArtificial SequenceSynthetic Primer
198caccctgaaa gaaagaaa 1819918DNAArtificial SequenceSynthetic
Peptide 199aagcctgaaa gaaagagt 1820018DNAArtificial
SequenceSynthetic Primer 200agaaatgaaa gaaagaga
1820118DNAArtificial SequenceSynthetic Primer 201ggtggtgaaa
gaaagaga 1820218DNAArtificial SequenceSynthetic Primer
202aggactgaaa gaaagagt 1820320DNAArtificial SequenceSynthetic
Primer 203gttcataacc gtggggctta 2020465DNAArtificial
SequenceSynthetic Primermisc_feature(1)..(2)Optional
phosphorothioate internucleoside
linkagemisc_feature(2)..(3)Optional phosphorothioate
internucleoside linkagemisc_feature(3)..(4)Optional
phosphorothioate internucleoside
linkagemisc_feature(62)..(63)Optional phosphorothioate
internucleoside linkagemisc_feature(63)..(64)Optional
phosphorothioate internucleoside
linkagemisc_feature(64)..(65)Optional phosphorothioate
internucleoside linkage 204ttgccccaca gggcagtaac ggcagacttc
tcctcaggag tcaggtgcac catggtgtct 60gtttg 6520522DNAArtificial
SequenceSynthetic Primer 205cttctccaca ggagtcaggt gc
2220629DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(2)n
= Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(29)..(29)Linked
to lys-lys-lys 206nntnttnctt ctccacagga gtcaggtgc
2920729DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(2)n
= Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(29)..(29)Linked
to lys-lys-lys 207nntnttnctt ctccacagga gtcaggtgc
2920829DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(2)n
= Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(29)..(29)Linked
to lys-lys-lys 208nntnttnctt ctccacagga gtcaggtgc
2920929DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(2)n
= Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(29)..(29)Linked
to lys-lys-lys 209nntnttnctt ctccacagga gtcaggtgc
2921025DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(1)..(2)n
= Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 210nntnttnctt ctccacagga gtcag 2521125DNAArtificial
SequenceSynthetic Peptide Nucleic Acidmisc_feature(1)..(1)Linked to
lys-lys-lysmisc_feature(1)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 211nntnttnctt ctccacagga gtcag 2521218DNAArtificial
SequenceSynthetic Primer 212tctccttaaa cctgtctt
1821325DNAArtificial SequenceSynthetic Peptide Nucleic
Acidmisc_feature(1)..(1)Linked to lys-lys-lysmisc_feature(3)..(4)n
= Pseudoisocytosinemisc_feature(6)..(6)n =
Pseudoisocytosinemisc_feature(7)..(8)Linked by three 8-amino-2, 6,
10-trioxaoctanoic acid, three 8-amino-3,6-dioxaoctanoic acid, or
three 6- aminohexanoic acid moleculesmisc_feature(25)..(25)Linked
to lys-lys-lys 213ttnntnttct ccttaaacct gtctt 252147DNAArtificial
Sequencesynthetic primermisc_feature(3)..(4)n =
Pseudoisocytosinemisc_feature(6)..(6)n = Pseudoisocytosine
214ttnntnt 72158DNAArtificial Sequencesynthetic
primermisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(4)..(4)n =
Pseudoisocytosinemisc_feature(7)..(7)n = Pseudoisocytosine
215tntnttnt 82168DNAArtificial Sequencesynthetic
primermisc_feature(2)..(2)n =
Pseudoisocytosinemisc_feature(5)..(5)n =
Pseudoisocytosinemisc_feature(7)..(8)n = Pseudoisocytosine
216tnttntnn 8
* * * * *
References